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Investigation of the zinc and manganese status of some stands of tsuga heterophylla in British Columbia 1991

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I N V E S T I G A T I O N OF THE Z I N C AND MANGANESE S T A T U S OF SOME STANDS TSUGA HETEROPHYLLA I N B R I T I S H C O L U M B I A by ROBERT G A D Z I O L A ( B . S c . F . , U n i v e r s i t y o f T o r o n t o , 1 9 8 1 ) A . T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF THE R E Q U I R E M E N T S FOR THE D E G R E E OF DOCTOR OF P H I L O S O P H Y i n THE F A C U L T Y OF G R A D U A T E S T U D I E S ( D e p a r t m e n t o f F o r e s t r y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H C O L U M B I A N o v e m b e r 1 9 9 1 0 R o b e r t G a d z i o l a , 1 9 9 1 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of \~c ^g,S^V^Y — The University of British Columbia Vancouver, Canada DE-6 (2/88) i i ABSTRACT Western hemlock has lower f o l i a r Zn and higher f o l i a r Mn c o n c e n t r a t i o n s compared to some other c o n i f e r s . E x i s t i n g f o l i a r d i a g n o s t i c norms f o r c o n i f e r s imply a Zn d e f i c i e n c y and p o s s i b l y a Mn t o x i c i t y i n many stands of western hemlock. This study was undertaken i n order to determine the s i g n i f i c a n c e of these f o l i a r l e v e l s i n the n u t r i t i o n of western hemlock. The n u t r i t i o n of hemlock was s t u d i e d using comparative n u t r i t i o n and f e r t i l i z e r s c r e e n i n g t r i a l s . The s c r e e n i n g t r i a l s c o n s i s t e d of treatments of Zn and Mn<applied as f o l i a r sprays and as s o i l t r e a t m e n t s . D i f f e r e n t methods of a p p l i c a t i o n were u t i l i z e d to determine i f f a c t o r s of the p l a n t such as uptake and/or t r a n s l o c a t i o n could account f o r the c h a r a c t e r i s t i c f o l i a r z i n c and manganese l e v e l s i n hemlock. In a d d i t i o n , a treatment was a p p l i e d c o n s i s t i n g of a complete f e r t i l i z e r without Zn and Mn. T h i s "complete-Zn-Mn" treatment was i n c l u d e d to i n v e s t i g a t e the p o s s i b i l i t y of a d d i t i o n a l n u t r i e n t d e f i c i e n c i e s and/or t o x i c i t i e s . In a comparison of t o t a l f o l i a r c o n c e n t r a t i o n s , hemlock had lower Zn compared to D o u g l a s - f i r , a m a b i l i s f i r and white p i n e . In c o n t r a s t , hemlock had higher Mn compared to Douglas- f i r , -amabilis f i r , white pine, red cedar and yellow cedar. A n a l y s i s of c e l l u l a r f r a c t i o n s of f o l i a g e produced two r e s u l t s . F i r s t , Zn accumulated i n the m i t o c h o n d r i a l f r a c t i o n and Mn accumulated i n the ribosomal and vacuolar f r a c t i o n , I r r e s p e c t i v e of the l e v e l of the treatment or the s p e c i e s . i i i Accumulation i n c e r t a i n f r a c t i o n s may i n d i c a t e a p h y s i o l o g i c a l need i n that f r a c t i o n or a t o l e r a n c e mechanism. Second, comparing hemlock to D o u g l a s - f i r , t o t a l Zn l e v e l s tend to be c o n s i s t e n t with l e v e l s i n d i f f e r e n t f r a c t i o n s , i n d i c a t i n g t h a t t o t a l l e v e l s may be an adequate i n d i c a t i o n of p h y s i o l o g i c a l l y a c t i v e l e v e l s . But comparing hemlock to D o u g l a s - f i r , t o t a l Mn l e v e l s are not c o n s i s t e n t with Mn l e v e l s i n d i f f e r e n t f r a c t i o n s , i n d i c a t i n g that t o t a l Mn l e v e l s may not be an adequate i n d i c a t i o n of p h y s i o l o g i c a l l e v e l s . In the f e r t i l i z e r s c r e e n i n g t r i a l s , n u t r i e n t uptake and growth responses were dependent upon the s i t e , the l e v e l of f e r t i l i z e r a p p l i c a t i o n , and the time s i n c e a p p l i c a t i o n . N u t r i e n t uptake and p o s i t i v e growth responses were obtained with f o l i a r treatments of Zn and s o i l treatments of Mn i n both the f i r s t year and second year f o l l o w i n g f e r t i l i z a t i o n . N u t r i e n t and growth responses to s o i l Zn treatments were delayed u n t i l the second year f o l l o w i n g f e r t i l i z a t i o n . A d d i t i o n a l evidence s u p p o r t i n g a Zn d e f i c i e n c y was i n d i c a t e d by a p o s i t i v e r e l a t i o n s h i p between f o l i a r Zn and height increment, evidence of r e t r a n s l o c a t i o n of Zn to new f o l i a g e i n the second year f o l l o w i n g f o l i a r Zn treatment, and the high ranking of Zn i n the v e c t o r a n a l y s i s from the "complete-Zn-Mn"treatment. P o s i t i v e growth responses to the "cooplete-Zn-Mn" treatment were obtained i n the f i r s t and second years f o l l o w i n g treatment. i v Ranking of n u t r i e n t response v e c t o r s using r e l a t i v e values i n d i c a t e d the e x i s t e n c e of other n u t r i e n t d e f i c i e n c i e s , besides Zn and Mn. Growth response, as measured by shoot increment r a t i o , was obtained p r i m a r i l y i n the second year a f t e r treatment with f o l i a r a p p l i c a t i o n s of Zn. Shoot increment r a t i o response occurred to s o i l Mn treatments i n the f i r s t year of treatment. For the "complete-Zn-Mn" t r e a t m e n t t h e r e was an i n c r e a s e i n shoot increment r a t i o i n both the f i r s t and second years f o l l o w i n g treatment. Height increment r a t i o i n c r e a s e d i n response to f o l i a r Zn a p p l i c a t i o n s i n the second year, and to s o i l Mn treatments i n the f i r s t year. F o l i a r Zn and f o l i a r N were p o s i t i v e l y c o r r e l a t e d with each o t h e r . F o l i a r Zn c o n c e n t r a t i o n s i n c r e a s e d as a r e s u l t of s o i l a p p l i c a t i o n s of Mn,- but a p p l i c a t i o n s of Zn had no e f f e c t on Mn uptake. T h e r e f o r e , there was no evidence In t h i s study to suggest t h a t low f o l i a r l e v e l s of Zn i n hemlock are due to a Mn antagonism. The only i n t e r a c t i o n obtained with the "complete-Zn -Mn" treatment was a synergism: i t caused an i n c r e a s e of f o l i a r Zn. Ingestad's n u t r i e n t r a t i o s were c a l c u l a t e d f o r the f o l i a r -levels -from the c o n t r o l and the "complete-Zn-Mn" treatments. V Comparing these r a t i o s to the optimum r e v e a l e d that most of the n u t r i e n t s were i n balance except f o r Fe and Mn. E x i s t i n g d i a g n o s t i c norms fo r Zn appear to adequately d e s c r i b e the Zn n u t r i t i o n of hemlock'. Response to f e r t i l i z a t i o n o ccurred with c o n t r o l f o l i a r Zn c o n c e n t r a t i o n s f o r hemlock being below the c r i t i c a l l e v e l of 15 pg g ~ x . D i a g n o s t i c norms f o r Mn need to be r e v i s e d . Response occurred-even though c o n t r o l f o l i a r Mn c o n c e n t r a t i o n s f o r hemlock were w e l l above the c r i t i c a l l e v e l of 25 ug g ~ v . T h e r e f o r e , t o t a l f o l i a r manganese may not be i n d i c a t i v e of the p h y s i o l o g i c a l manganese s t a t u s of hemlock. These r e s u l t s f o r hemlock are d i s c u s s e d i n l i g h t of e x i s t i n g knowledge from the l i t e r a t u r e r e g a r d i n g the n u t r i e n t s t r a t e g y of metal t o l e r a n t p l a n t s and low n u t r i e n t adapted p l a n t s . v i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v i LIST OF TABLES x LIST OF FIGURES . . . . x i i i LIST OF APPENDICES xv ACKNOWLEDGEMENTS x v i i CHAPTER 1. INTRODUCTION 1 CHAPTER 2 . LITERATURE REVIEW . . 4 A. N u t r i t i o n a l C h a r a c t e r i s t i c s of Western Hemlock 4 B. Zinc and Manganese L e v e l s i n P l a n t s 9 C. N u t r i t i o n a l D i f f e r e n c e s Between P l a n t s . 14 D. D e f i c i e n c y and T o x i c i t y L e v e l s f o r Zinc and Manganese. . 20 E. Nutrient-Growth Models 22 1. C l a s s i c a l Growth Response Curve.. 22 2 . - Ingestad's N u t r i e n t Flux Density Model....24 F. Diagnosis of N u t r i e n t Status and Requirements ..29 1. P l a n t and S o i l A n a l y s e s . . . . ..... 29 2. F o l i a r A n a l y s i s 30 (a) Background 30 (b) P h y s i o l o g i c a l and E m p i r i c a l Basis of F o l i a r A n a l y s i s 31 (c) N u t r i e n t Diagnosis Using F o l i a r A n a l y s i s 33 ( i ) T o t a l A n a l y s i s with S i n g l e N u t r i e n t s . 33 v i i Page ( i i ) T o t a l A n a l y s i s using N u t r i e n t Balances 40 ( i i i ) P h y s i o l o g i c a l A c t i v e F r a c t i o n s . . . . 44 G. CONCLUSIONS .... 48 CHAPTER 3. METHODS AND MATERIALS.. 51 A. S i t e D e s c r i p t i o n 51 1. L o c a t i o n of Study Areas 51 2. Stand C h a r a c t e r i s t i c s 52 3. S o i l C h a r a c t e r i s t i c s . . . . . . . .52 B. Experimental Design 54 C. F e r t i l i z e r Treatments... ....59 D. F i e l d Sampling 60 E. Chemical A n a l y s i s 63 F. Scanning E l e c t r o n Microprobe 65 G. S o i l Sample P r e p a r a t i o n and A n a l y s i s . 66 H. Measurement of F e r t i l i z e r Response... ...67 I. S t a t i s t i c a l A n a l y s i s . . . 69 CHAPTER 4. RESULTS ..71 A. Comparative N u t r i t i o n . . . . . . . . . . ...71 1. T o t a l L e v e l s .71 2. E x t r a c t a b l e Z i n c and Manganese 71 3. C e l l u l a r F r a c t i o n s of F o l i a g e 73 B. F e r t i l i z a t i o n Experiments .. 79 1. N u t r i e n t and Growth Responses 79 (a) Z i n c 83 ( i ) F o l i a r Z i n c Treatments 83 v i i i Page ( i i ) S o i l Zinc Treatments 87 ( i i i ) Comparison of F o l i a r Versus S o i l Treatments 90 (b ) Manganese 90 ( i ) F o l i a r Manganese Treatments ...90 ( i i ) S o i l Manganese Treatments 93 (c) Complete-Zn-Mn 97 ( i ) N u t r i e n t and Growth Responses ... 97 2. Shoot Increment R a t i o . 103 3. Height Increment Ratio 108 C. R e t r a n s l o c a t i o n . . . . . . . . . . ....108 D. Nutrient-Growth. I n t e r a c t i o n s , 114 E. N u t r i e n t I n t e r a c t i o n s 117 1. I n t e r a c t i o n s with Zinc 117 (a) Manganese 117 -; (b) Nitrogen 117 2. I n t e r a c t i o n s with Manganese 122 (a) Z i n c 122 F. R e l a t i o n s h i p of Response to S i t e 126 CHAPTER 5. DISCUSSION 131 A. Comparative N u t r i t i o n 131 1. T o t a l L e v e l s 131 2. E x t r a c t a b l e Zn and Mn 133 3. C e l l u l a r F r a c t i o n s 134 B. F e r t i l i z a t i o n Experiments 135 1. N u t r i e n t and Growth Responses 135 (a) Zinc 136 i x Page ( i ) Comparison of F o l i a r Versus S o i l Treatments ..136 ( i i ) Z i n c Tolerance 140 (b) Manganese..... -....143 ( i ) Comparison of F o l i a r Versus S o i l Treatments .144 ( i i ) T o x i c i t y L e v e l s 146 ( i l l ) Manganese Requirements .......148 ( i v ) Manganese T o l e r a n c e . . . .. 149 C. Comparison and C o n s i d e r a t i o n of Adequate L e v e l s 150 1. I n d i v i d u a l N u t r i e n t s . . 150 2. N u t r i e n t Balance 156 D. R e t r a n s l o c a t i o n . . . . .159 E. N u t r i e n t I n t e r a c t i o n s 160 F. F o l i a r A p p l i c a t i o n of Zinc i n Forestry.......165 CHAPTER 6. CONCLUSIONS ..166 LITERATURE CITED. »v . . . . . . . .173 APPENDICES vl88 X LIST OF TABLES 1. Comparison of s u r f a c e s o i l and f o r e s t f l o o r p r o p e r t i e s under D o u g l a s - f i r and western hemlock stands from c o a s t a l versus Cascade s i t e s . 7 2. T i s s u e m i c r o n u t r i e n t l e v e l s i n P a c i f i c Northwest c o n i f e r s 10 3. F o l i a r Zn/Mn l e v e l s f o r v a r i o u s t r e e s p e c i e s o c c u r r i n g i n the same stands l o c a t e d i n the i n t e r i o r of B.C . .13 4. L i n e a r c o r r e l a t i o n s between growth response and c u r r e n t needle N c o n c e n t r a t i o n , N content and dry weight i n the two years f o l l o w i n g f e r t i l i z a t i o n . ... ; .34 5. General r e p r e s e n t a t i o n of changes i n t o t a l content, y i e l d and c o n c e n t r a t i o n as a f f e c t e d by imposed treatments ... ....38 6. Chemical c h a r a c t e r i s t i c s of the s o i l p r o f i l e s . . 55 7. T r e a t m e n t - l e v e l s used i n the f e r t i l i z e r t r i a l s with the number used i n the t e x t and i t s corresponding c o n t r o l . . . . . 5 6 8. T o t a l f o l i a r Zn, Mn and Fe, water-soluble Zn and Mn, and a c t i v e Fe f o r d i f f e r e n t s p e c i e s i n the same sta n d s . . . . . . . . 72 9. Equations and R a values f o r the height increment versus f o l i a r shoot-' per mass r e l a t i o n s h i p s . ...... . . . . 82 10. F o l i a r z i n c n u t r i e n t response and f o l i a r mass per shoot response to f o l i a r a p p l i c a t i o n s of z i n c i n the f i r s t and second years f o l l o w i n g treatments ..84 11; F o l i a r z i n c and f o l i a r mass per shoot response to s o i l a p p l i c a t i o n s of z i n c i n the second year . . 88 12. F o l i a r manganese and f o l i a r mass per shoot response to s o i l treatments of manganese. ........94 13. N u t r i e n t response to complete-Zn-Mn treatment... 98 14. Shoot increment r a t i o growth response to z i n c ...104 15. Shoot increment r a t i o response to s o i l a p p l i c a t i o n s of manganese i n the f i r s t year (1986) on s i t e s 4 and 5. 106 x i Table Page 16. Shoot increment r a t i o response to the complete-Zn-Mn treatment 107 17. Height increment r a t i o response.......... 109 18. Change i n f o l i a r z i n c c o n c e n t r a t i o n of c u r r e n t year's f o l i a g e with time and age of f o l i a g e f o l l o w i n g treatment f o r the low z i n c f o l i a r treatment (4) on s i t e s 1 and 2 I l l 19. Change i n f o l i a r z i n c c o n c e n t r a t i o n s of c u r r e n t year's f o l i a g e with time and age of f o l i a g e f o l l o w i n g treatment f o r the high z i n c f o l i a r treatment (5) on s i t e s 1 and 2 .112 20. Change i n f o l i a r manganese c o n c e n t r a t i o n of c u r r e n t year's f o l i a g e with time and age of f o l i a g e f o l l o w i n g treatment f o r the manganese s o i l treatment (3) on s i t e s 1 and 2 113 21. Equations and R a values f o r the height increment vesus f o l i a r z i n c r e l a t i o n s h i p s . 116 22. F o l i a r manganese response to s o i l and f o l i a r a p p l i c a t i o n s of z i n c i n the f i r s t year (1986) on s i t e 5 118 23. Equations and H' values f o r the f o l i a r n i t r o g e n versus f o l i a r z i n c r e l a t i o n s h i p s . . . . . . 120 24. F o l i a r z i n c response to s o i l and f o l i a r manganese a p p l i c a t i o n s ...123 25. F o l i a r z i n c response i n the f i r s t year (1986) to s o i l a p p l i c a t i o n s of manganese on s i t e 5 124 26. Summary of n u t r i e n t and- growth responses to f o l i a r z i n c treatments 127 27. Summary of n u t r i e n t and growth responses to s o i l z i n c treatments. 128 28. Summary of n u t r i e n t and growth responses to s o i l manganese treatments ....129 '29. Summary of n u t r i e n t and growth responses to the complete -Zn-Mn treatment.. 130 30. S o i l data f o r a l l s i t e s . 132 31. E f f e c t of s o l u t i o n pH on m i c r o n u t r i e n t c o n c e n t r a t i o n s i n D o u g l a s - f i r ari*d western hemlock r o o t s and needles 142 x i i Table Page 32. Ingestad's f o l i a r n u t r i e n t r a t i o s f o r the complete-Zn -Mn and c o n t r o l treatments on s i t e s 4 and 5 f o r the years 1986 and 1987.. . . . 157 x i i i LIST OF FIGURES Figure Page 1. F a c t o r s of the p l a n t which a f f e c t p l a n t n u t r i t i o n 15 c 2. The three models of p l a n t - s o i l r e l a t i o n s h i p s . . . . . . . . . . . . 19 3. R e l a t i o n s h i p between p l a n t growth and t i s s u e n u t r i e n t c o n c e n t r a t i o n s . 23 4. The r e l a t i o n s h i p between the e x t e r n a l n u t r i e n t supply and r e l a t i v e growth r a t e ... .. ... 26 5. Schematic -representation of a d e t a i l e d mechanistic model of t r e e growth.......... 32 6. Vector method f o r the i n t e r p r e t a t i o n of n u t r i e n t -growth response data using n u t r i e n t c o n c e n t r a t i o n , n u t r i e n t content and dry mass of needles 37 7. Example of a boundary l i n e . . . . ...39 8. Diagramatic r e p r e s e n t a t i o n of crop response to a number of l i m i t i n g f a c t o r s . . . . . . 41 9. L o c a t i o n of the study p l o t s i n the Lower Hainland.......52 10* Z i n c c o n c e n t r a t i o n s i n d i f f e r e n t c e l l u l a r f r a c t i o n s from the c u r r e n t year's f o l i a g e (1986) of the c o n t r o l treatment and high f o l i a r Zn treatment from s i t e 5 .75 11. > Manganese c o n c e n t r a t i o n s i n d i f f e r e n t c e l l u l a r f r a c t i o n s from the c u r r e n t year's f o l i a g e (1986) of the c o n t r o l treatment and high s o i l Mn treatment from s i t e 5 76 12. Manganese c o n c e n t r a t i o n s i n d i f f e r e n t c e l l u l a r f r a c t i o n s from the c u r r e n t year's f o l i a g e i n 1987 of d i f f e r e n t s p e c i e s on s i t e 5 .....77 13. Z i n c c o n c e n t r a t i o n s i n d i f f e r e n t c e l l u l a r f r a c t i o n s from the c u r r e n t year's f o l i a g e i n 1987 of d i f f e r e n t s p e c i e s on s i t e 5 78 14. S c a t t e r p l o t s of height increment versus the f o l i a r mass per-shoot f o r s i t e 1 i n t h e f i r s t and second years f o l l o w i n g treatment .81 15. Vector diagrams of growth response to f o l i a r a p p l i e d z i n c i n the f i r s t year on s i t e 2, i n the second year on s i t e 2, i n the second year on s i t e 3, and i n the f i r s t year on s i t e 5 86 x i v F i gure Page 16. Vector diagram of the second year g r o w t h r e s p o n s e to s o i l a p p l i e d z i n c on s i t e 4 f o r f o l i a r z i n c ..89 17. N u t r i e n t e f f i c i e n c y of f o l i a r Zn versus s o i l Zn treatments i n s u p p l y i n g the p l a n t with Zn .91 18. Vector diagrams of the second year growth response to f o l i a r a p p l i e d manganese on s i t e s 4 and 5 f o r f o l i a r manganese. ....92 19. Vector diagram of the growth response to s o i l a p p l i e d Mn f o r f o l i a r Mn i n the second year on s i t e 4 and i n the f i r s t year on s i t e 5... 96 20. Vector diagram of the f i r s t year growth response to the complete-Zn-Mn treatment on s i t e 4. 101 21. Vector diagrams of the f i r s t and second year growth response to the complete-Zn-Mn treatment on s i t e S 102 22. S c a t t e r p l o t s of height increment versus c u r r e n t year's f o l i a r z i n c l e v e l s i n the f i r s t year and i n the second year s i t e 4 115 23. S c a t t e r p l o t s of f o l i a r n i t r o g e n versus f o l i a r z i n c i n the f i r s t and second years from s i t e 1 119 24. S c a t t e r p l o t of t o t a l s o i l n i t r o g e n versus e x t r a c t a b l e s o i l z i n c both from the f o r e s t f l o o r f o r a l l s i t e s . . . . . 121 25. Vector diagram of f i r s t year n u t r i e n t response of z i n c to s o i l treatments of manganese from s i t e 5... 125 26. P o s s i b l e pathways of s o l u t e movement through the leaf..145 27. Ribbon model of a s i n g l e z i n c f i n g e r domain (ADRla) i n c o r p o r a t i n g t e t r a h e d r a l c o o r d i n a t i o n of z i n c by c y s t i n e (C) and h i s t i d i n e (H) 164 XV LIST OF APPENDICES Appendix Page A . l S i t e and S o i l D e s c r i p t i o n of S i t e 1 ....188 A.2 S i t e and S o i l D e s c r i p t i o n of S i t e 2 .....190 A.3 S i t e and S o i l D e s c r i p t i o n of S i t e 3 192 A.4 S i t e and S o i l D e s c r i p t i o n of S i t e 4... . . .. .194 A. 5 S i t e and S o i l D e s c r i p t i o n of S i t e 5 196 B. 1 M o d i f i e d Parkinson and A l l e n D i g e s t i o n f o r P l a n t T i s s u e A n a l y s i s . 198 B.2 N i t r i c A c i d D i g e s t i o n f o r A n a l y s i s of Copper and Iron i n F o l i a g e . . 199 B.3 Procedure f o r the Determination of Sulphate-Sulphur i n F o l i a g e 200 B.4 Determination of A c t i v e Iron i n F o l i a g e . 201 B.5 Determination of E x t r a c t able Z i n c . . 202 B. 6 Determination-of E x t r a c t a b l e Manganese from F o l i a g e . . . . 203 C. Comparison of F o l i a r Zn, Mn, and Fe C o n c e n t r a t i o n s Using AA and ICP....... ... 204 D. Formulae Used to Convert F o l i a r Zn and Mn C o n c e n t r a t i o n s Measured on the AA to Corresponding ICP Values > 205 E. Comparison of the- Recovery of F o l i a r N u t r i e n t s from AA and ICP 206 F. P r e p a r a t i o n and A n a l y s i s of C e l l u l a r F r a c t i o n s from F o l i a g e 208 G. F i x a t i o n and Embedding Procedure 209 H. The Mehlich 3 S o i l E x t r a c t i o n Method 210 I. F o l i a r n u t r i e n t g u i d e l i n e s f o r the i n t e r p r e t a t i o n of n u t r i t i o n a l s t a t u s 212 J . F o l i a r N u t r i e n t Data f o r the C e l l u l a r F r a c t i o n s . . . . . . . . 2 1 7 K . l S c a t t e r p l o t of the 1986 height increment versus the 1986 f o l i a r mass per shoot f o r s i t e 2. .... .219 x v i Appendix Page K.2 S c a t t e r p l o t of the 1986 height Increment versus the 1986 f o l i a r mass per shoot f o r s i t e 3. 220 K.3 S c a t t e r p l o t of the 1986 height increment versus the 1986 f o l i a r mass per shoot f o r s i t e 4... ....221 K.4 S c a t t e r p l o t of the 1 9 8 7 h e i g h t I n c r e m e n t versus the 1987 f o l i a r mass per shoot f o r s i t e 4.. 222 K.5 S c a t t e r p l o t of t h e 1 9 8 6 h e i g h t increment versus the 1986 f o l i a r mass per shoot f o r s i t e 5 223 R.6 S c a t t e r p l o t of the 1987 height increment versus the 1987 f o l i a r mass per shoot f o r s i t e 5 224 "L. F o l i a r N u t r i e n t Data from the F e r t i l i z a t i o n T r i a l s . . . . . 225 M.i F o l i a r N u t r i e n t Data from S i t e 1 f o r the Study of Zn and Mn R e t r a n s l o c a t i o n with time and age...............264 M.2 F o l i a r N u t r i e n t Data from S i t e 2 f o r the Study of Zn and Mn R e t r a n s l o c a t i o n with time and age 265 M.I S c a t t e r p l o t of f i r s t year t o t a l height increment i n 1986 versus f i r s t year f o l i a r z i n c i n 1986 f o r s i t e 5. .266 N.2 S c a t t e r p l o t of second year t o t a l height increment i n 1987 versus f o l i a r z i n c l e v e l s i n 1987 f o r s i t e 5 267 .0.1 S c a t t e r p l o t of f o l i a r n i t r o g e n versus f o l i a r z i n c f o r c u r r e n t year's f o l i a g e i n 1985 f o r s i t e 2. ...268 0.2 S c a t t e r p l o t of f o l i a r n i t r o g e n versus f o l i a r z i n c f o r - c u r r e n t year's f o l i a g e i n 1986 f o r s i t e 2. 269 0.3 S c a t t e r p l o t of f o l i a r n i t r o g e n versus f o l i a r z i n c f o r c u r r e n t year's f o l i a g e i n 1986 f o r s i t e 3 ..270 0.4 S c a t t e r p l o t of f o l i a r n i t r o g e n versus f o l i a r z i n c f o r c u r r e n t year's f o l i a g e i n 1986 f o r s i t e 4. 271 0.5 S c a t t e r p l o t -of f o l i a r n i t r o g e n versus f o l i a r z i n c f o r c u r r e n t year's f o l i a g e i n 1987 f o r s i t e 4. .272 0.6 S c a t t e r p l o t of f o l i a r n i t r o g e n versus f o l i a r z i n c f o r c u r r e n t year's- f o l i a g e i n 1986 f o r s i t e 5....... 273 0.7 S c a t t e r p l o t of f o l i a r n i t r o g e n versus f o l i a r z i n c f o r c u r r e n t year's f o l i a g e i n 1987 f o r s i t e 5. ..274 x v i i ACKNOWLEDGEMENTS I am a p p r e c i a t i v e to my s u p e r v i s o r y Dr. T. M; B a l l a r d f o r p r o v i d i n g the i n i t i a l i d e a f o r t h i s t h e s i s , and f o r h i s support and e f f o r t i n b r i n g i n g t h i s work to i t s f r u i t i o n . I am a l s o g r a t e f u l to the other members of my Su p e r v i s o r y Committee: Dr. A. A. Bomke, Dr. Ki K l i n k a , and Dr. 6. F. Weetman f o r the time and e f f o r t they took to review and provide c o n s t r u c t i v e c r i t i c i s m of t h i s t h e s i s . The c o n t r i b u t i o n of Dr. R. J . Zasos k i i s acknowledged f o r the c o n t r i b u t i o n he provided as the E x t e r n a l Examiner; In producing t h i s t h e s i s I wish to acknowledge the c o n t r i b u t i o n s made by Mr. R. C a r t e r , Mrs. R. Lowe, Ms. E. Wolterson, and Mr. B. Von S p i n d l e r . I would a l s o l i k e to thank the s t a f f of -the M i s s i o n Tree Farm ( M i s s i o n , B.C.), and the U.B.C. Research F o r e s t (Maple Ridge, B.C.) f o r p r o v i d i n g me with the f a c i l i t i e s to e s t a b l i s h f e r t i l i z e r t r i a l s . I would l i k e to thank the v a r i o u s o r g a n i z a t i o n s which provided me with the f i n a n c i a l support which allowed me to attend graduate s c h o o l and conduct t h i s r e s e a r c h . These were the Canadian F o r e s t r y S e r v i c e , the Natur a l Sciences and Engineering Research' C o u n c i l of Canada, F l e t c h e r Challenge of Canada L t d . , the F a c u l t y of F o r e s t r y , and the Department of S o i l S c i e n c e . x v i i i In a d d i t i o n , the f r i e n d s h i p s provided by f e l l o w students and s t a f f i n the- Department of S o i l Science and F a c u l t y of F o r e s t r y deeply e n r i c h e d my l i f e . F i n a l l y , I am t h a n k f u l to my f a m i l y f o r their- l o v e , support and encouragement. I ded i c a t e t h i s t h e s i s i n memory of my Father., Leon Peter G a d z i o l a . 1 CHAPTER 1. INTRODUCTION The s p e c i e s Tsuga heterophylla (Raf.) Sarg. (western hemlock) c h a r a c t e r i s t i c a l l y has lower f o l i a r z i n c (Zn) and higher f o l i a r manganese (Mn) c o n c e n t r a t i o n s r e l a t i v e to some other c o n i f e r s p e c i e s throughout B r i t i s h Columbia and the United S t a t e s P a c i f i c Northwest. C u r i o s i t y about t h i s phenomena motivated t h i s r e s e a r c h . The p a t t e r n s of f o l i a r Zn and Mn l e v e l s i n hemlock are of p r a c t i c a l s i g n i f i c a n c e i n f o r e s t r y . Attempts to i n c r e a s e the p r o d u c t i v i t y of hemlock with n i t r o g e n f e r t i l i z e r s have met with v a r i a b l e and i n c o n s i s t e n t r e s u l t s . T h i s has lead to i n t e r e s t i n the s t a t u s of other n u t r i e n t s i n hemlock such as Zn and Mn. . In comparison to suggested c r i t i c a l f o l i a r l e v e l s f o r some B.C. c o n i f e r s , hemlock o f t e n has f o l i a r - Z n . l e v e l s which f a l l i n t o the low to p o s s i b l y d e f i c i e n t zone (being l e s s than the c r i t i c a l f o l i a r Zn l e v e l of 15 ug g ~ x ) , and has f o l i a r Mn l e v e l s which f a l l i n t o the high to p o s s i b l y t o x i c range (being higher then the c r i t i c a l f o l i a r Mn l e v e l of 25 ug g ~ * ) . T h e r e f o r e , the f o l i a r Zn and Mn c o n c e n t r a t i o n s c h a r a c t e r i s t i c of hemlock suggest that Zn i s at p o s s i b l y d e f i c i e n t and Mn i s at p o s s i b l y t o x i c l e v e l s . Up to the present time the p a t t e r n s of f o l i a r Zn and Mn c o n c e n t r a t i o n s of hemlock have been d e s c r i b e d but no work has been done on t h e i r s i g n i f i c a n c e i n the n u t r i t i o n of hemlock. 2 T h e r e f o r e , the o b j e c t i v e of t h i s study was to i n v e s t i g a t e the s i g n i f i c a n c e of the p a t t e r n of f o l i a r Zn and Mn c o n c e n t r a t i o n s i n the n u t r i t i o n of hemlock. The s p e c i f i c q u e s t i o n s asked were: Is Zn sometimes d e f i c i e n t and i s Mn sometimes t o x i c f o r the growth of hemlock? Are the lower f o l i a r Zn c o n c e n t r a t i o n s due to a Mn antagonism? Are the lower f o l i a r Zn and higher f o l i a r Mn c o n c e n t r a t i o n s of hemlock compared to some other c o n i f e r s due to f a c t o r s of the p l a n t such as uptake and/or t r a n s l o c a t i o n or f a c t o r s of the s o i l such as f e r t i l i t y ? Are there a d d i t i o n a l n u t r i e n t d e f i c i e n c i e s i n hemlock? A comprehensive l i t e r a t u r e review was made to l e a r n what was a l r e a d y known about hemlock n u t r i t i o n , Zn and Mn l e v e l s i n p l a n t s * p l a n t f a c t o r s a f f e c t i n g n u t r i t i o n , d e f i c i e n c y and t o x i c i t y l e v e l s f o r Zn and Mn, nutrient-growth r e l a t i o n s h i p s , and methods of n u t r i e n t d i a g n o s i s . Two approaches were taken i n t h i s r e s e a r c h . The f i r s t s t ep i n v o l v e d a comparison of Zn and Mn n u t r i t i o n among s e v e r a l c o n i f e r s p e c i e s , to check the premise f o r t h i s study. This l e d to another q u e s t i o n : although p l a n t s may have d i f f e r e n t t o t a l f o l i a r Zn and Mn c o n c e n t r a t i o n s do they have s i m i l a r p h y s i o l o g i c a l l e v e l s ? A review of the l i t e r a t u r e suggests some p o s s i b l e mechanisms, and some experimental work ( e x t r a c t a b l e and a c t i v e n u t r i e n t l e v e l s , and f o l i a r Zn and Mn d i s t r i b u t i o n s between c e l l u l a r f r a c t i o n s ) was d i r e c t e d to t h i s q u e s t i o n . The second step i n v o l v e d the use of f e r t i l i z e r s c r e e n i n g t r i a l s , 3 using f o l i a r and s o i l treatments of Zn and Mn, and a s o i l a p p l i c a t i o n of a "complete-Zn-Mn" treatment. By measuring n u t r i e n t and growth responses, i n f e r e n c e s c o u l d be made about d e f i c i e n c i e s and t o x i c i t i e s . N u t r i e n t r e t r a n s l o c a t i o n , n u t r i e n t i n t e r a c t i o n s , and nutrient-growth r e l a t i o n s h i p s could a l s o be examined using data from the f e r t i l i z e r t r i a l s . Since the p a t t e r n s of Zn and Mn f o l i a r c o n c e n t r a t i o n s are found over a wide area of hemlock's range, the s i t e s s e l e c t e d f o r the f e r t i l i z e r t r i a l s d i d not cover a wide range of ecosystem c o n d i t i o n s . In a d d i t i o n , t h e enormity of c o l l e c t i n g s e v e r a l hundred samples, each of which would be s u b j e c t e d to s e v e r a l chemical a n a l y s e s , and the requirement of having s i t e s e a s i l y a c c e s s i b l e from the U n i v e r s i t y of B r i t i s h Columbia to f a c i l i t a t e the work, l i m i t e d ' the number of s i t e s which c o u l d be i n v e s t i g a t e d . These c o n s t r a i n t s o b v i a t e d using data and c o n c l u s i o n s to g e n e r a l i z e about hemlock i n the r e g i o n as a whole. However, the s i t e s e l e c t i o n proved adequate f o r i t s purpose: i t succeeded i n f i n d i n g hemlock stands low i n Zn and high i n Mn s u i t a b l e f o r t e s t i n g hypotheses about d e f i c i e n c y , t o x i c i t y and antagonism. 4 CHAPTER 2. LITERATURE REVIEW A. N u t r i t i o n a l C h a r a c t e r i s t i c s of Western Hemlock Western hemlock occurs i n f i v e b i o g e o c l i m a t i c zones of B. C. I t grows as a climax s p e c i e s i n the C o a s t a l and I n t e r i o r Western Hemlock zones. In the C o a s t a l D o u g l a s - F i r zone, i t develops- as a climax s p e c i e s only i n su b h y d r i c h a b i t a t s . In the suba l p i n e Mountain Hemlock and Engelmann Spruce-Subalpine F i r zones, the s h o r t v e g e t a t i v e season i s the major o b s t a c l e which l i m i t s 'the d i s t r i b u t i o n and growth of western hemlock ( K r a j i n a et al. 1982). Western hemlock i s commonly a s s o c i a t e d with s t r o n g l y p o d z o l i z e d s o i l s and these s o i l s c h a r a c t e r i s t i c a l l y have a mor humus l a y e r which has a low r a t e of m i n e r a l i z a t i o n . T h i s suggests that hemlock i s t o l e r a n t of a c i d i c s o i l s and r e q u i r e s lower q u a n t i t i e s of n u t r i e n t s compared to some other t r e e s ( K r a j i n a er al. 1982). The p r o d u c t i v i t y of hemlock was found by Lowe and K l i n k a (1981) to have a lower negative c o r r e l a t i o n with the percent y i e l d of s o i l l i p i d s of the humus l a y e r as compared to D o u g l a s - f i r . L i p i d s accumulate to high l e v e l s i n - s o i l s where b i o l o g i c a l a c t i v i t y i s i n h i b i t e d (Lowe and K l i n k a 1981), which i n d i c a t e s a negative r e l a t i o n s h i p between the abundance of s o i l l i p i d s and m i n e r a l i z a t i o n . The lower c o r r e l a t i o n between the amount of s o i l l i p i d s and the p r o d u c t i v i t y of hemlock suggests that hemlock i s l e s s s e n s i t i v e to low r a t e s of decomposition, 5 m i n e r a l i z a t i o n and n u t r i e n t r e l e a s e compared to D o u g l a s - f i r (Lowe and K l i n k a 1981). The p r o d u c t i v i t y of hemlock was a l s o p o s i t i v e l y c o r r e l a t e d to the pyrophosphate-extractable Fe+Al (PFeAl) and carbon content i n the B h o r i z o n (TCB). Both t h e PFeAl and TCB are I n d i c e s of the extent and i n t e n s i t y of podzol formation. T h i s helps to e x p l a i n the occurrence of hemlock on p o d z o l i c s o i l s . Hemlock r e g e n e r a t i o n f l o u r i s h e s on r o t t e n l o g s , stumps or mineral s o i l exposed on t r a i l s or on mounds and p i t s c r e a t e d by windthrown t r e e s ( C h r i s t y et al. 1982; Stewart 1989). The or g a n i c l a y e r i s f r e q u e n t l y a substratum f o r hemlock even above the more b a s e - r i c h , high pH s o i l l a y e r s i n which t h i s t r e e does •not regenerate ( K r a j i n a et al. 1982). Rotten wood i s an important s u b s t r a t e - f o r m y c o r r h i z a l a s s o c i a t i o n s of moist hemlock h a b i t a t types i n the northern Rockies (Harvey er al. 1979). More than 95% of the a c t i v e primary r o o t s of hemlock have been found to be m y c o r r h i z a l ( G i l l - and Lavender 1983). Since mycorrhizae - have been found to enhance; n u t r i e n t uptake i n many s p e c i e s , t h e i r abundance on hemlock r o o t s suggests that mycorrhizae p l a y an important r o l e i n hemlock n u t r i t i o n . During the f i r s t season, s e e d l i n g s are able to s u r v i v e on r o t t e n wood without mycorrhizae; however, mycotrophy i s advantageous f o r growth (Harvey e r al. 1979). Two-year-old s e e d l i n g s growing on mineral s o i l had gr e a t e r growth than those growing on the r o t t e n wood s u b s t r a t e . The e f f e c t of s u b s t r a t e on growth disappeared i n the t h i r d year ( C h r i s t y et al. 1982). Roots of s e e d l i n g s which o r i g i n a l l y e s t a b l i s h e d on r o t t e n wood e v e n t u a l l y extended t h e i r r o o t s i n t o mineral s o i l and the o r i g i n a l s u b s t r a t e disappeared. The n u t r i t i o n a l source then s h i f t s from woody s u b s t r a t e to mineral s o i l ( C h r i s t y er al. 1982). Hemlock occurs- on s o i l s which c h a r a c t e r i s t i c a l l y have low pH and a pH-dependent c a t i o n exchange c a p a c i t y (CEC); o f t e n low base s a t u r a t i o n and abundant A l on the s o i l exchange complex; high o r g a n i c matter contents; and the presence of s e s q u i o x i d e s and/or " a n d i c " s o i l p r o p e r t i e s (Ryan 1983). T h i s i s evident when one compares s o i l p r o p e r t i e s f o r hemlock f o r e s t s versus Douglas- f i r - f o r e s t s (Table 1 ) . Consequently, s o l u b l e A l and Mn are high (Toy 1984), and n i t r o g e n i s predominantly i n the ammonium (NH**) form- (Haynes 1986). Anderson er al. (-1982) found t h a t n i t r i f i c a t i o n i n two c o a s t a l Washington s o i l s under hemlock stands was n e g l i g i b l e . T h i s was a l s o found f o r f o r e s t f l o o r and mineral s o i l s a s s o c i a t e d with hemlock t r e e s i n northwestern Washington (Turner and Franz 1985).. T h e r e f o r e , the n i t r o g e n n u t r i t i o n of hemlock appears to be adapted to the ammonium form. N u t r i e n t s t u d i e s by K r a j i n a er al. (1973), and van den Driessche (1971, 1976) tend to support t h i s o b s e r v a t i o n . Ryan er al. (1986a, 1986b) i n v e s t i g a t e d the t o l e r a n c e of s e e d l i n g s of D o u g l a s - f i r , western hemlock, and western redcedar to s o l u t i o n a c i d i t y and A l c o n c e n t r a t i o n i n s o l u t i o n c u l t u r e s . Western hemlock s u r v i v e d and t h r i v e d i n a c i d s o l u t i o n of pH 3 but the other s p e c i e s had g r e a t e r m o r t a l i t y and reduced growth (Ryan 7 Table I. Comparison of s u r f a c e s o i l and f o r e s t f l o o r p r o p e r t i e s under second growth D o u g l a s - f i r (A) and western hemlock (B) stands from c o a s t a l (W) versus Cascade (E) s i t e s (from Zas o s k i et al. (1986)). Region A B Surface S o i l 0-15 cm PH E 5.1 4.5 W 4.9 4.4 Exchangeable Ca t i o n s (meq per 100-8 •)• Ca E 3.8 1.7 W 3.0 1.2 Kg E 0.8 0.36 W 0.96 0.73 K E 0.41 0.27 W 0.38 0.32 CEC E 26.0 35.1 W 37.1 47.3 Base S a t u r a t i o n (X) E 20.4 7.2 W 11.4 4.8 A v a i l a b l e P (pg g E 97.0 44.0 W 51.0 15.0 T o t a l P (|ig E 1070 809 W 13 70 954 A v a i l a b l e S (ug g -*) E 7.7 9.9 W 9.5 10.2 T o t a l N (%) E 0.16 0.31 W 0.31 0.45 8 Table 1. (con c l u d e d ) . T o t a l C (%) E 4.7 8.4 W 8.3 10.9 C:N Ratio E 30.3 28.5 W 27.2 24.4 Fo r e s t F l o o r T o t a l Mass (kg ha"*) T o t a l N (%) T o t a l C (X) E 21700 29800 W 18700 27000 E 0.95 1.05 W 1.05 1.02 E 39.1 41.5 W 39.7 43.4 C:N Ratio E 38.7 W 39.0 38.0 42.9 9 et al. 1986a). Both western hemlock and western red cedar were found to be e s p e c i a l l y t o l e r a n t of a c i d - A l c o n d i t i o n s with a s o l u t i o n pH of 3.5 and at the highest A l treatment of 100 ug g ~ l (Ryan er al. 1986b). Aluminium a d v e r s e l y a f f e c t e d the t i s s u e c o n c e n t r a t i o n s of Ca and Mg. The a b i l i t y of western hemlock to grow i n a c i d - A l c o n d i t i o n s i s suggested to be r e l a t e d to t h i 6 s p e c i e s ' p h y s i o l o g i c a l t o l e r a n c e of excess H-cations i n s o l u t i o n and low t i s s u e requirements of Ca and Mg (Ryan et al. 1986a, 1986b). The s p e c i f i c p h y s i o l o g i c a l or b i o c h e m i c a l A l t o l e r a n c e mechanisms f o r hemlock have not been i n v e s t i g a t e d . In a mature mixed s u b a l p i n e stand, T. aertensiana and A. amabilis had the h i g h e s t c o n c e n t r a t i o n of A l i n the f i n e r o o t component r e l a t i v e to a l l t i s s u e s analyzed (Vogt et al. 1987). It was hypothesized that accumulation i n the r o o t s i s an e f f e c t i v e mechanism f o r a v o i d i n g Al t o x i c i t y . The l a r g e root biomasses of these s u b a l p i n e stands allow f o r l a r g e amounts of A l to be taken up and immobilized i n r o o t s . The high root turnover i n these stands may be a r e s u l t of r o o t senescence o c c u r r i n g i n response to high A l accumulation. Root senescence would be an e f f e c t i v e mechanism f o r removing A l from the b i o l o g i c a l component (Vogt er al. 1987b). B. Z i n c and Manganese L e v e l s i n P l a n t s The c o n c e n t r a t i o n s of Zn and Mn i n the f o l i a g e of v a r i o u s t r e e s p e c i e s i n the P a c i f i c Northwest are l i s t e d i n Tables 2 and 3. Hemlock tends to have lower f o l i a r Zn and higher f o l i a r Mn 10 Table 2. Ti s s u e m i c r o n u t r l e n t l e v e l s (|ig g~M i n P a c i f i c Northwest c o n i f e r s . Species Age Type L o c a t i o n Zn Mn Source D o u g l a s - f i r 13-49 f i e l d Van. I s . B.C. 17-35 452 -758 1 1 outdoors S e a t t l e , WA 11-28 125 -785 2 4 p l a n t Hoquiam, WA 21-32 390 -580 3 7 p l a n t Pack For. WA 21 4 20 f i e l d Puget Sound WA 14-31 350 -2010 5 30 f i e l d C o a s t a l B.C. 11-18 174 -465 6 30 f i e l d C o a s t a l B.C. 23 121 6 5-32 p l a n t C o a s t a l B.C. 13 292 7 5-32 p l a n t C o a s t a l B.C. 8 316 ,. . 7 60-73 f i e l d C o a s t a l B.C. 18 186 7 plugs nursery S e a t t l e , WA 35 8 S i t k a spruce 1 outdoors plugs nursery Western red cedar plugs nursery 27 f i e l d 25 f i e l d Ponderosa pine plugs nursery 7 p l a n t S e a t t l e , WA S e a t t l e , WA S e a t t l e , WA Coast OR, WA, B.C. I n t e r i o r OR, WA S e a t t l e , WA Pack For., 40 WA 31-68 98-403 59 24 13-26 69-383 21^-48 102-368 64 2 8 8 9 9 8 4 11 Table 2 (continued) Western hemlock 1 outdoors S e a t t l e , 33-36 140-619 2 WA plugs nursery S e a t t l e , 45 8 WA 23-39 f i e l d Cascades, 13-24 885-1213 10 OR 23-39 cont. C o a s t a l 7-15 617-764 10 OR 23-39 f i e l d Cascades 15-23 736-1087 10 OR 23-39 f e r t . C o a s t a l 9-15 434-586 10 OR 60-150 f i e l d Van. I s . 3-14 1580-198 1 B.C. 4 p l a n t Hoquiam, 10-19 480-930 3 WA 1-2 l a t h Olympla, 0.1-3 249-696 11 WA 20-30 f i e l d Cascades 16-20 1200-190 12 WA 20-30 f i e l d C o a s t a l 17-20 1000-110 12 WA 6-10 f i e l d Western 17-19 616-1922 13 WA 13-15 p l a n t O z e t t , WA - 7-16 14 70 f i e l d Gold R i v e r , 5 1804 15 B.C. 70 f i e l d Kaprino I, 3 1017 15 B.C. 72 f i e l d Kaprino I I , I 1263 15 B.C. 52 f i e l d I s l a n d Hwy, 5 1493 15 B.C. 70 f i e l d Beaver Lake , 6 1720 15 B.C. 71 f i e l d Rupert Main » 5 1626 15 B.C. 30 f i e l d B r i t t a i n 7 1187 15 R i v e r , B.C. 5.9 1114 15 6.6 753 15 6.4 880 15 7.4 1043 15 7.7 930 15 8.8 1127 15 7.8 811 15 12 Table 2 <concluded) Western white pine - 20 f i e l d Sub-alpine f i r Puget 26-62 Sound, WA Lodgepole pine White spruce Douglas f i r Western red cedar Spruce h y b r i d f i e l d f i e l d f i e l d f i e l d f i e l d f i e l d I n t e r i o r , B.C. I n t e r i o r , B.C. I n t e r i o r , B.C. I n t e r i o r , B.C. I n t e r i o r , B.C. I n t e r i o r , B.C. 16-64 33-71 37-76 13-27 8-12 141-1800 369-1175 245-747 154-1025 274-2244 92-384 33-66 225-637 16 16 16 16 16 16 Sources 1. Beaton er al. 1965 2. Rollwagen 1981 3. Porada 1987 4. G r e e n l e a f - J e n k i n s 1985 5. Z a s o s k i et al. 1977 6. C a r t e r et al. 1984 7. C a r t e r er al. 1986 8. Z a s o s k i et al. 1984 9. Radwan and Ha r r i n g t o n 1987 10. G i l l and Lavender 1983 11. Radwan and DeBell 1980a 12. Radwan and DeBell 1980b 13. Z a s o s k i er al. 1990 14. Z a s o s k i et al. 1990 15. C a r t e r unpublished 16. 'Ballard unpublished 13 Table 3. F o l i a r Zn/Mn l e v e l s (|ig g~M f o r v a r i o u s t r e e s p e c i e s o c c u r r i n g i n the sane stands l o c a t e d i n the i n t e r i o r of B.C. (from B a l l a r d ( p e r s o n a l communication) 1). Species P l o t s Western hemlock 3/2225 Western red cedar Douglas f i r 14/697 Lodgepole pine 50/662 White spruce Spruce h y b r i d 1/825 2/850 5/1850 2/732 11/358 11/100 11/280 14/338 53/227 61/474 Species P l o t s Western hemlock 3/1625 3/2425 3/1775 1/1125 5/975 Western red cedar 12/316 11/384 Spruce h y b r i d 47/427 Species P l o t s Western hemlock 1/1175 5/1275 Western red cedar 8/180 D o u g l a s - f i r 18/379 1. T. M. B a l l a r d . P r o f e s s o r , F a c u l t y of Forestry/Department of S a i l S c i e n c e , U n i v e r s i t y of B r i t i s h Columbia. Unpublished Data 1984. 14 c o n c e n t r a t i o n s compared to other s p e c i e s , not onl y i n d i f f e r e n t stands (Table 2 ) , but a l s o i n the same stands (Table 3 ) . In a mixed subalpine stand of A. amabilis and T. mertensiana, the two s p e c i e s were found to have c l e a r d i f f e r e n c e s i n t h e i r a b i l i t y to accumulate s p e c i f i c elements from the s o i l (Vogt et al. 1987a). D i f f e r e n t i a l ' n u t r i e n t - l e v e l s have a l s o been found between other wild p l a n t s p e c i e s on the same s i t e s ( G e r l o f f er al. 1966), and between a g r i c u l t u r a l ^ p l a n t s of d i f f e r e n t s p e c i e s (Gladstones and Loneragan 1970; C o l l a n d e r 1941) and v a r i e t i e s when s u p p l i e d with the same amounts of n u t r i e n t s (Brown et al. 1972). T h i s i n d i c a t e s that d i f f e r e n t s p e c i e s of p l a n t s have d i f f e r e n t t o l e r a n c e s and requirements for' n u t r i e n t s , and that f a c t o r s of the p l a n t s themselves a f f e c t n u t r i e n t uptake. C. N u t r i t i o n a l .Differences' between P l a n t s There are a number of reasons f o r d i f f e r e n c e s i n n u t r i t i o n between s p e c i e s and genotypes which are summarized i n Fig u r e 1. These are r e l a t e d to uptake, t r a n s p o r t and u t i l i z a t i o n i n the p l a n t (Marschner 1986). Both uptake and growth are assumed to be c o n t r o l l e d by cyto p l a s m i c pools through feedback and s u b s t r a t e supply, r e s p e c t i v e l y . Consequently, s p e c i e s d i f f e r e n c e s i n uptake and growth might be r e l a t e d to c e l l u l a r compartmentation of n u t r i e n t s (Chapin 1988). Species adapted to d i f f e r e n t s o i l f e r t i l i t i e s g e n e r a l l y d i f f e r i n the d i s t r i b u t i o n of n u t r i e n t s among v a r i o u s chemical f r a c t i o n s (Chapin 1988). Species may 15 (I) Nutrient Efficiency (1) (2) a) Demand on cellular level a) Root-shoot transport (long distance) b) Utilization within the shoot (eg. retran8locatlon) W Transport within the root (short distance) c) Compartmentatlon/bindlng- form within the roots (II) Acquisition of (1) Root morphology a) Roots themselves . i) inherent il) response to deficiency b) Mycorrhizae (eg. secretions < (2) Root physiology and biochemistry a) Affinity of the uptake system (Km) b) Threshold concentration (Cmln) c) Modifications of the rhlzosphere I) Passive (eg. cation-anlon uptake) ii) Active response to deficiency chelating, reducing compounds, protons) F i g u r e 1. F a c t o r s of the p l a n t which a f f e c t p l a n t n u t r i t i o n (from Marschner (1986)). 16 d i f f e r i n the compartmentation of n u t r i e n t s i n t o s p e c i f i c p l a n t p a r t s , chemical f r a c t i o n s , or c e l l u l a r compartments (Chapin- 1988). F o l i a r c o n c e n t r a t i o n s may r e f l e c t these d i f f e r e n c e s (Bowen 1981), or some of the same f r a c t i o n s may have s i m i l a r - n u t r i e n t c o n c e n t r a t i o n s f o r the same p h y s i o l o g i c a l processes. Hemlock tends to have lower n u t r i e n t requirements compared to other c o n i f e r s ( K r a j i n a et al. 1982). P l a n t s adapted to low n u t r i e n t c o n d i t i o n s have a growth r a t e which i s r e l a t i v e l y i n s e n s i t i v e to v a r i a t i o n i n the r a t e of n u t r i e n t supply (Chapin 1988). During p e r i o d s of high n u t r i e n t a v a i l a b i l i t y there would be accumulation of vacuolar s t o r e s ( l u x u r y consumption). Luxury consumption of n u t r i e n t s b u f f e r s the p l a n t from v a r i a t i o n i n e x t e r n a l n u t r i e n t supply (Chapin 1988). There i s a tendency towards s t a b l e i o n i c composition of- the cytoplasm (Glass and S i d d i q i 1984; Leigh and Jones 1986). P l a n t s can a f f e c t n u t r i e n t uptake by a f f e c t i n g the pH of the r h i z o s p h e r e . There are d i f f e r e n c e s i n the rh i z o s p h e r e pH among p l a n t s p e c i e s growing i n the same s o i l (Harsc'hner 1986). Hemlock has a higher r a t i o of H* r e l e a s e / MH** uptake than D o u g l a s - f i r . T h i s suggests hemlock may not onl y t o l e r a t e a c i d c o n d i t i o n s but would tend to c r e a t e a c i d i t y i n the rhiz o s p h e r e (Bygiewicz et al. 1984). Hemlock had lower mean ammonium uptake r a t e s f o r both m y c o r r h l z a l and nonmycorrhizal r o o t s compared to D o u g l a s - f i r . However, m y c o r r h l z a l r o o t s enhanced ammonium uptake r a t e s i n hemlock (Rygiewicz et al. 1984). 17 The Corn of n i t r o g e n used by a p l a n t can have an i n f l u e n c e on the p l a n t ' s o v e r a l l n u t r i t i o n . P l a n t s with a n n o n i u i n u t r i t i o n tend to absorb c a t i o n s i n excess of anions (N being the element o f t e n absorbed i n the l a r g e s t amounts), with a net e f f l u x of H* i n t o the r h i z o s p h e r e . As a consequence of ammonium n u t r i t i o n there i s a decrease i n the uptake of c a t i o n s compared to that observed with n i t r a t e n u t r i t i o n . T h i s may be a t t r i b u t e d to i o n i c c o m p e t i t i o n with ammonium or with the excreted H*" i o n s (Haynes 1986) . The r e d u c t i o n i n c a t i o n uptake with ammonium n u t r i t i o n may be a mechanism of p l a n t t o l e r a n c e to A l and Mn on a c i d s o i l s . In g e n e r a l , ammonium i n h i b i t s the p l a n t ' s uptake of Mn and Al (Haynes 1986). Ammonium n u t r i t i o n has two other consequences. F i r s t l y , b i n d i n g of free- ammonium must take place with o r g a n i c compounds i n order to t o l e r a t e the high ammonium l e v e l s ( Kirkby and Hughes 1970). Secondly, ammonium p l a n t s must ensure a s y n t h e s i s of org a n i c anions independently of n i t r a t e r e d u c t i o n as a means of compensating f o r the inadequate supply of anions. The sm a l l e r q u a n t i t y of anions would hinder the t r a n s f e r of c a t i o n s to the shoots. It i s through an a c t i v e s y n t h e s i s of org a n i c a c i d s t h a t p l a n t s could have an e q u i v a l e n t or b e t t e r growth when they depend on ammonium as a n i t r o g e n source ( S a l s a c e r al. 1987) . 18 Baker (1981), I d e n t i f i e d three n u t r i e n t models of p l a n t - s o i l r e l a t i o n s . These are the accumulator, the excluder and the i n d i c a t o r ( F i g u r e 2 ) . Accumulators are p l a n t s where metals are concentrated i n above-ground p l a n t p a r t s from low or high s o i l l e v e l s (Baker 1981). Excluders are p l a n t s where metal c o n c e n t r a t i o n s i n the shoots are maintained constant and low over a wide range of s o i l c o n c e n t r a t i o n s , by d i f f e r e n t i a l uptake and t r a n s p o r t , up to a c r i t i c a l s o i l value above which the mechanism breaks down and u n r e s t r i c t e d t r a n s p o r t r e s u l t s . I n d i c a t o r s are p l a n t s where uptake and t r a n s p o r t of metals to the shoot are re g u l a t e d so that i n t e r n a l c o n c e n t r a t i o n r e f l e c t s e x t e r n a l l e v e l s (Baker 1981). In both the accumulators and the e x c l u d e r s , the mechanisms of t o l e r a n c e are l a r g e l y ' i n t e r n a l ' i n that there i s a c t i v e d e t o x i f i c a t i o n of metal i o n s . I t i s the s i t e s of d e t o x i f i c a t i o n which d i f f e r , being l a r g e l y w i t h i n the root i n e x c l u d e r s and i n the shoots i n accumulators (Baker 1981). D e t o x i f i c a t i o n may r e s u l t from c e l l - w a l l b i n d i n g , a c t i v e pumping of ions i n t o vacuoles, complexing by org a n i c a c i d s and p o s s i b l y by s p e c i f i c m e tal-binding p r o t e i n , enzymatic adaptations and e f f e c t s on membrane p e r m e a b i l i t y (Baker 1987). Ectomycorrhizae may p l a y a r o l e i n metal e x c l u s i o n by r e s t r i c t i n g uptake or through accumulation. T a y l o r (1987) made a d i s t i n c t i o n between i n t e r n a l t o l e r a n c e and e x c l u s i o n based upon the s i t e of metal 19 Accumulator Indicator Plant Plant Soil F i g u r e 2. The t h r e e models of p l a n t - s o i l r e l a t i o n s h i p s . The axes r e p r e s e n t n u t r i e n t c o n c e n t r a t i o n s (from Baker (1981)). 20 d e t o x i f i c a t i o n o r i m m o b i l i z a t i o n , e i t h e r i n the symplasm ( i n t e r n a l ) or apoplasm ( e x c l u s i o n ) of r o o t s . E x c l u s i o n of a metal may be by i m m o b i l i z a t i o n i n the c e l l w a l l , exudation of c h e l a t e s or org a n i c a c i d s from r o o t s , a redox b a r r i e r at the plasma membrane, or a pH b a r r i e r at the plasma membrane ( T a y l o r 1987). D. Deficiency- and T o x i c i t y L e v e l s f o r Zn and Mn The f o l i a r n u t r i e n t c o n c e n t r a t i o n s tend to be an i n t e g r a t i o n of a l l the s o i l and p l a n t f a c t o r s which a f f e c t n u t r i e n t a v a i l a b i l i t y to the p l a n t . Therefore, they serve as an i n d i c a t o r of p l a n t n u t r i e n t a v a i l a b i l i t y . The c r i t i c a l Mn d e f i c i e n c y l e v e l found i n general f o r most p l a n t f o l i a g e ranges from 15 to 25 ug g~* with the s u f f i c i e n t l e v e l being 20-300 ug g-*- (Kabata-Pendias and Pendias 1984). T o x i c i t y to Mn g e n e r a l l y occurs at a c o n c e n t r a t i o n above about 500 ug g - 1 (Kabata-Pendias and Pendias 1984); t o x i c i t y to Mn at f o l i a r c o n c e n t r a t i o n s o f over 1,000 ug g~* Is common(National Academy of Sciences 1973). There i s abundant evidence which suggests t h a t Mn l e v e l s found i n p l a n t s are not r e f l e c t i v e of p h y s i o l o g i c a l requirements. Species as d i v e r s e as sugarbeets and wheat have a c r i t i c a l d e f i c i e n c y l e v e l i n the range of 10-20 ug g~x ( C l a r k s o n 1988). In Vacciniua vitis-idaea, p l a n t s having f o l i a r Mn l e v e l s of 6,800 21 to 12,300 ug g~x had s i m i l a r r a t e s of p h o t o s y n t h e s i s , dry n a t t e r p r o d u c t i o n and number of l e a v e s as p l a n t s c o n t a i n i n g 18 to 1,500 ug g-*- of Mn ( M i l l e r 1987). In t r e e s p e c i e s f r o n the temperate f o r e s t s of C e n t r a l Japan, Mn c o n c e n t r a t i o n s of the shoots vary between s p e c i e s by a f a c t o r of 180 (Marschner 1988). In a d d i t i o n , l a r g e d i f f e r e n c e s have been found between b a r l e y genotypes i n Mn e f f i c i e n c y . T h e r e f o r e , d i f f e r e n c e s i n Mn c o n c e n t r a t i o n s between s p e c i e s growing on the same s o i l probably have l i t t l e to do with d i f f e r e n c e s i n Mn requirements (Clarkson 1988) or u t i l i z a t i o n i n the p l a n t s (Marschner 1988). They are probably due to d i f f e r e n c e s i n Mn a c q u i s i t i o n from the s o i l (Marschner 1988). These d i f f e r e n c e s may p a r t i a l l y r e f l e c t the extent to which s p e c i e s are able to a c i d i f y the rhizosphere ( C l a r k s o n 1988) or produce Mn r e d u c i n g o r g a n i c r o o t exudates. Memon and Yatazawa (1982) e x t r a c t e d w a t e r - s o l u b l e Mn from Mn accumulator p l a n t s . More than 70% of the t o t a l Mn was water- s o l u b l e . R e s u l t s of e l e c t r o n probe x-ray m i c r o a n a l y s i s revealed that the p o r t i o n of Mn which was w a t e r - i n s o l u b l e was contained i n the c e l l w a l l s . The c r i t i c a l Zn d e f i c i e n c y l e v e l f o r p l a n t s i s from 10-20 ug g " 1 , with the l e v e l of s u f f i c i e n c y being from 27-150 ug g ~ l , and the t h r e s h o l d of t o x i c i t y at 100-400 ug g ~ x (Kabata-Pendias and Pendias 1984). There are some r e p o r t s that o n l y a p o r t i o n of the t o t a l l e v e l of z i n c i s p h y s i o l o g i c a l l y a c t i v e . Water-soluble Zn has been examined as an i n d i c a t i o n of the Zn s t a t u s of the 22 p l a n t . Cakmak and Marschner (1987) found water-soluble z i n c to be a s u i t a b l e i n d i c a t o r of z i n c n u t r i t i o n a l s t a t u s i n g e n e r a l . T h i s was because of the c l o s e c o r r e l a t i o n between water-soluble z i n c and v i s u a l Zn d e f i c i e n c y symptoms, l e v e l s of c h l o r o p h y l l , s u p e r o x i d e disrautase, and membrane p e r m e a b i l i t y . Rahimi and Schropp (1984) found water - s o l u b l e z i n c to be an i n d i c a t o r of the Zn n u t r i e n t s t a t u s of the p l a n t because of i t s r e l a t i o n s h i p to the a c t i v i t y of c a r b o n i c anhydrase. When one t r i e s to make an accounting of a l l the z i n c , there i s a d i s c r e p a n c y between t o t a l and i d e n t i f i a b l e z i n c (Hewitt 1983). Up to 60% of the p l a n t z i n c has been accounted f o r i n i t s i d e n t i f i a b l e forms i n p r o t e i n s (Hewitt 1983). E. Nutrient-Growth Models 1. C l a s s i c a l P l a n t Growth Curve The c l a s s i c a l p l a n t nutrient-growth model i s an e m p i r i c a l r e l a t i o n s h i p d e s c r i b e d by a curve of d i m i n i s h i n g r e t u r n s . The curve may represent a r e l a t i o n s h i p between p l a n t growth and t i s s u e n u t r i e n t c o n c e n t r a t i o n s ( F i g u r e 3 ), between p l a n t growth and e i t h e r s o i l n u t r i e n t c o n c e n t r a t i o n s or f e r t i l i z e r a d d i t i o n s . The curve c o n s i s t s of four zones: d e f i c i e n t (A-B), s u f f i c i e n t (B- C), luxury (C-D) and t o x i c i t y (D-E). The d e f i c i e n t zone may be d i s t i n g u i s h e d from the adequate zone by the c r i t i c a l n u t r i e n t 23 F i g u r e 3 . R e l a t i o n s h i p b e t w e e n p l a n t g r o w t h and t i s s u e n u t r i e n t c o n c e n t r a t i o n s . 24 c o n c e n t r a t i o n . T h i s i s the n u t r i e n t c o n c e n t r a t i o n that corresponds to 90% of maximum y i e l d . Other m o d i f i c a t i o n s of the curve by Dow and Roberts (1982) have a c r i t i c a l n u t r i e n t range r a t h e r than a c r i t i c a l n u t r i e n t c o n c e n t r a t i o n (B-C). T h i s r e l a t i o n s h i p has been expressed mathematically using the M i t s c h e r l i c h model, q u a d r a t i c and e x p o n e n t i a l models, and i n v e r s e polynomials and h y p e r b o l i c models (Walworth and Sumner 1988). T h i s t r a d i t i o n a l model of p l a n t growth-nutrient r e l a t i o n s h i p s has c e r t a i n t h e o r e t i c a l problems. F i r s t l y , t h i s type of r e l a t i o n s h i p i s e m p i r i c a l and does not l e a d to r e s u l t s of g e n e r a l a p p l i c a t i o n (Landsberg 1986). From the growth curve one attempts to d e f i n e c r i t i c a l f o l i a r n u t r i e n t c o n c e n t r a t i o n s by • e s t a b l i s h i n g r e l a t i o n s h i p s between f i n a l y i e l d , or growth increment over a p e r i o d , and n u t r i e n t c o n c e n t r a t i o n i n the f o l i a g e , measured at the end of that p e r i o d . These lead to a second problem i n that the r e s u l t s are h i g h l y v a r i a b l e because the t i s s u e n u t r i e n t s t a t u s (x) at any time ( t ) a f f e c t s the growth r a t e at that time (dy/dt) (Equation 1) (Landsberg 1986). dy/dt - f ( x ) (I) where: y = growth t = time x = n u t r i e n t c o n c e n t r a t i o n 2. Ingestad's N u t r i e n t Flux Density Model 25 An a l t e r n a t i v e model of n u t r i t i o n and growth, the n u t r i e n t f l u x d e n s i t y model has been formulated and demonstrated by Ingestad ( F i g u r e 4 ) . The b a s i c premise of the model i s that i t i s the r a t e of n u t r i e n t supply to the r o o t s or r e l a t i v e a d d i t i o n r a t e (RA) (amount of n u t r i e n t added per u n i t of time and u n i t of n u t r i e n t present i n the p l a n t ) which i s the d r i v i n g v a r i a b l e of growth w i t h i n the sub-optimum range up to and i n c l u d i n g the optimum. Since p l a n t growth i s e x p o n e n t i a l with time i n the sub- optimum range, a n u t r i e n t alone or n u t r i e n t s i n f i x e d p r o p o r t i o n s must be s u p p l i e d i n e x p o n e n t i a l l y i n c r e a s i n g amounts ( r e l a t i v e a d d i t i o n r a t e RA) corresponding to the e x p o n e n t i a l growth of the p l a n t (So) (equation 2 ) , and t h e r e f o r e the r e l a t i v e n u t r i e n t uptake r a t e (Ru) w i l l be p r o p o r t i o n a l to RA and Ro (Ingestad and Lund 1986). Ro = C R A (2) where: Ro • l/W(dW/dt) RA = l/W(dM/dt) c ••*» constant of p r o p o r t i o n a l i t y W - t o t a l p l a n t mass M = n u t r i e n t c o n c e n t r a t i o n t • time 27 Under f i e l d c o n d i t i o n s the n u t r i e n t f l u x d e n s i t y (amount of n u t r i e n t s a v a i l a b l e per u n i t of time and u n i t of area) corresponds to RA (Ingestad 1987). The n u t r i e n t f l u x d e n s i t y may be regarded as n u t r i e n t flow which e n t e r s the p l a n t , s i m i l a r to energy flow (Ingestad er al. 1981). A constant r e l a t i v e growth r a t e and constant i n t e r n a l n u t r i e n t c o n c e n t r a t i o n can only be maintained where the n u t r i e n t supply to the r o o t s i n c r e a s e s i n p r o p o r t i o n to the r e l a t i v e growth. The curve ( F i g u r e 4) i s not continuous l i k e the c l a s s i c a l response curve but c o n s i s t s of a sub-optimum range (the s t r a i g h t l i n e below the s a t u r a t i o n p o i n t ) which i s r e l a t e d to RA, and a supra-optimum range (the curve to the r i g h t of the s a t u r a t i o n p o i n t ) (Ingestad 1982). In the c l a s s i c a l response model where a constant amount of n u t r i e n t s i s added per u n i t of time the amount added becomes uptake r e s t r i c t i n g . T h i s i s due to the f a c t that the added amounts become l e s s and l e s s s u f f i c i e n t i n r e l a t i o n to the requirements of both the i n t e r n a l c o n c e n t r a t i o n and r e l a t i v e growth. There are two consequences. F i r s t l y , the d e f i c i e n c y l e v e l i s overestimated because the c l a s s i c a l response curve i s the r e s u l t of changing i n t e r n a l n u t r i e n t s t a t e . T h e r e f o r e , p l a n t s can be grown having much lower t i s s u e c o n c e n t r a t i o n s when steady s t a t e n u t r i t i o n i s maintained. Secondly, the p o t e n t i a l maximum growth i s underestimated using the c l a s s i c a l response curve because of i n s u f f i c i e n t n u t r i e n t a d d i t i o n r a t e s to maintain the growth r a t e . D e f i c i e n c y symptoms occur dur i n g the l a g phase when the growth r a t e of a p l a n t i s a d j u s t i n g from a higher to a 28 lower RA r e s u l t i n g i n a decrease i n the i n t e r n a l n u t r i e n t c o n c e n t r a t i o n . The symptoms disappear once a new steady s t a t e i s formed between RA and Ro r e s u l t i n g i n a s t a b l e i n t e r n a l n u t r i e n t c o n c e n t r a t i o n (Ingestad 1982). At the optimum RA the n u t r i e n t requirement i s s a t u r a t e d and a f u r t h e r i n c r e a s e of RA does not i n c r e a s e the Ro. The e x t e r n a l n u t r i e n t c o n c e n t r a t i o n i n c r e a s e s because the RA i s not matched by a corresponding i n c r e a s e i n the Ru (Ingestad 1982). I t i s suggested t h a t steady s t a t e n u t r i t i o n i s the c h a r a c t e r i s t i c s i t u a t i o n under n a t u r a l c o n d i t i o n s because growth a d j u s t s to the n u t r i t i o n a l resources of the s i t e (Ingestad 1982). Agren (1985) has put f o r t h the n u t r i e n t p r o d u c t i v i t y model i n which the r e l a t i v e growth rate i s p r o p o r t i o n a l to the amount of a n u t r i e n t i n the p l a n t with the n u t r i e n t p r o d u c t i v i t y being the p r o p o r t i o n a l i t y f a c t o r . The n u t r i e n t p r o d u c t i v i t y i s a constant f o r a given s p e c i e s under f i x e d environmental c o n d i t i o n s (Agren 1985). Agren (1988) has produced a s i n g l e f o r m u l a t i o n which r e l a t e s growth to the content of s e v e r a l n u t r i e n t s . The p l a n t growth r a t e i s p r o p o r t i o n a l to the n u t r i e n t content minus a given minimum c o n c e n t r a t i o n of the n u t r i e n t (Equation 3 ) . The p r o p o r t i o n a l i t y f a c t o r , the n u t r i e n t p r o d u c t i v i t y , and the minimum c o n c e n t r a t i o n are s p e c i e s - s p e c i f i c . Ro s P A{niin(c n |c n |«>pt) - c n,m«.n) (3) = PnH where: P« i s n u t r i e n t p r o d u c t i v i t y = (l/M)(dW/dt) 29 = (l/NMRo) Re i s p l a n t growth r a t e • dW/dt Cn.nin i s the minimum c o n c e n t r a t i o n of a n u t r i e n t t h a t oust be present before any growth i s r e a l i z e d . Cn.opt i s the upper c o n c e n t r a t i o n of a n u t r i e n t above which no f u r t h e r growth response i s obt a i n e d . c n i s n u t r i e n t c o n c e n t r a t i o n N s (nin(Cn}Cn|ept) ~ Cn,aln) H i s the optimum c o n c e n t r a t i o n l/N i s n u t r i e n t e f f i c i e n c y F. Diagnosis of N u t r i e n t S tatus and Requirements 1. Plant and S o i l A n a l y s i s The d i a g n o s i s of the n u t r i t i o n a l s t a t u s of t r e e s may be performed i n s e v e r a l ways through p l a n t a n a l y s i s or s o i l a n a l y s i s . The aim of d i a g n o s t i c p l a n t a n a l y s i s i s to use some c h a r a c t e r i s t i c of the p l a n t which i s r e f l e c t i v e of p l a n t n u t r i e n t s t a t u s . P l a n t a n a l y s i s may i n v o l v e examination of v i s u a l symptoms of d e f i c i e n c i e s or t o x i c i t i e s . However, i t would be d e s i r a b l e to make a d i a g n o s i s before the n u t r i t i o n a l problem has manifested i t s e l f m o r p h o l o g i c a l l y . Other methods of pl a n t 30 a n a l y s i s i n c l u d e chemical a n a l y s i s of the f o l i a g e , buds, phloem, xylem sap, wood, bark, r o o t s , and l i t t e r . D i a g n o s t i c s o i l n u t r i e n t a n a l y s i s commonly attempts to use chemical e x t r a c t a n t s to remove that f r a c t i o n of n u t r i e n t s which may be p l a n t a v a i l a b l e . An advantage of s o i l a n a l y s i s i s that the n u t r i e n t s t a t u s of the s o i l c ould be determined p r i o r to p l a n t a t i o n establishment (Mead 1984). However, t h i s method r e q u i r e s the c a l i b r a t i o n of the e x t r a c t e d n u t r i e n t l e v e l s with p l a n t n u t r i t i o n and growth a f t e r determining the chemical e x t r a c t i n g s o l u t i o n which can give a u s e f u l index of n u t r i e n t a v a i l a b i l i t y . 2. F o l i a r A n a l y s i s a. Background In t h i s study f o l i a r a n a l y s i s has been used as a d i a g n o s t i c t o o l . S e v e r a l reviews of f o l i a r a n a l y s i s have been prepared e x p l a i n i n g i t s theory, sampling, i n t e r p r e t a t i o n and l i m i t a t i o n s . These have been by van den Driessche (1974), Lavender (1970), Everard (1973), Walworth and Sumner (1988), Bates (1971), and B a l l a r d and C a r t e r (1986). P i o n e e r i n g work i n f o l i a r a n a l y s i s was done by Lundegardh (1951, 1947, 1943), Chapman (1941), Thomas (1937, 1945), Thomas and Mack (1941, 1944), U l r i c h (1943), Moser (1940) and S c a r s e t h (1943). I 31 b. P h y s i o l o g i c a l and E m p i r i c a l Basis of F o l i a r A n a l y s i s The aim of d i a g n o s t i c f o l i a r a n a l y s i s c a l i b r a t i o n i s to determine the r e l a t i o n s h i p between p l a n t performance and f o l i a r composition, thus e n a b l i n g the use of the l a t t e r as an index of the n u t r i e n t s t a t u s of a p l a n t . It does not r e v e a l anything about why the p l a n t may have a d e f i c i e n c y , s u f f i c i e n c y or t o x i c i t y of a n u t r i e n t . There i s both a p h y s i o l o g i c a l and an e m p i r i c a l b a s i s f o r f o l i a r a n a l y s i s . Figure 5 presents a somewhat mechanistic model of t r e e growth based on p h y s i c a l / p h y s i o l o g i c a l p r o c e s s e s . I t i l l u s t r a t e s the r e l a t i o n s h i p between p l a n t n u t r i e n t s t a t u s , l e a f area and t r e e growth. The amount of f o l i a g e i s an important f a c t o r i n determining the amount of s o l a r r a d i a t i o n i n t e r c e p t e d by the canopy. The amount of s o l a r r a d i a t i o n i n t e r c e p t e d i n t u r n i s an important f a c t o r a f f e c t i n g t r e e growth. F e r t i l i z a t i o n with a g r o w t h - l i m i t i n g n u t r i e n t i n a non-closed c o n i f e r o u s stand can be expected to i n c r e a s e growth by i n c r e a s i n g needle biomass ( l e a f s i z e , area, number), thus i n c r e a s i n g the p h o t o s y n t h e t i c c a p a c i t y ( L i n d e r and Rook 1984). Increases i n l e a f area a f t e r f e r t i l i z a t i o n are r e l a t e d to i n c r e a s e s i n r a t e s of p h o t o s y n t h e s i s per l e a f area and stem wood pro d u c t i o n ( L i n d e r and Rook 1984). T h i s has been found f o r Scots p i n e , D o u g l a s - f i r and Pinus nigra ( L i n d e r and Rook 1984). Vose and A l l e n (1988) found that l e a f area index (LAI) i n c r e a s e d up to 60* f o l l o w i n g H f e r t i l i z a t i o n on two K - d e f i c i e n t s i t e s and that 32 F i g u r e 5 . S c h e m a t i c m o d e l o f t r e e g r o w t h r e p r e s e n t a t i o n o f a d e t a i l e d ( f r o m L a n d s b e r g ( 1 9 8 6 ) ) . m e c h a n i s t i c 33 stemwood pr o d u c t i o n was p o s i t i v e l y r e l a t e d to LAI. Leyton (1956) detected an i n c r e a s e i n the c u r r e n t l e a f s i z e which preceded an i n c r e a s e i n height growth i n the f o l l o w i n g growing season, induced by changes i n s o i l f e r t i l i t y . E m p i r i c a l r e l a t i o n s h i p s have a l s o been found i n t e r n s of c o r r e l a t i o n s between f o l i a r parameters and subsequent t r e e growth (Table 4 ) . c. Plant N u t r i e n t Diagnosis Using F o l i a r A n a l y s i s i . T o t a l A n a l y s i s with S i n g l e N u t r i e n t s F o l i a r a n a l y s i s i n v o l v e s e v a l u a t i o n of the f o l i a g e i n terms of n u t r i e n t c o n c e n t r a t i o n , n u t r i e n t content, f o l i a r mass, on a f o l i a r area b a s i s or using n u t r i e n t r a t i o s . Using combinations of these parameters, s e v e r a l procedures have been used for n u t r i e n t d i a g n o s i s . The r e l a t i o n s h i p of growth to n u t r i t i o n based on the c l a s s i c a l p l a n t growth curve i s the f i r s t method which may be used f o r p l a n t n u t r i e n t d i a g n o s i s . The f i r s t stage of d i a g n o s i s i n v o l v e s i n f e r r i n g the e x i s t e n c e of one or more n u t r i e n t d e f i c i e n c i e s or t o x i c i t i e s through the use of v i s u a l symptoms, by comparing f o l i a r l e v e l s to those p l a n t s having s u p e r i o r growth, or by comparing f o l i a r l e v e l s to c r i t i c a l l e v e l s f o r the s p e c i e s ( a p p l y i n g an e x i s t i n g c a l i b r a t i o n ) or f o r other s p e c i e s . Since the r e l a t i o n s h i p i n p a r t of the d e f i c i e n t zone i s q u a s i - l i n e a r , l i n e a r r e g r e s s i o n a n a l y s i s has sometimes been a p p l i e d f o r 34 Table 4. L i n e a r c o r r e l a t i o n s ( r a ) between growth response and c u r r e n t needle N c o n c e n t r a t i o n (NX), N content (Nc) and u n i t dry weight (Wt) i n the two years f o l l o w i n g f e r t i l i z a t i o n . A l l stands were N d e f i c i e n t (from Timmer (1979)). Response Response Season NX Nc Wt Species Parameter Period (year) Leader 2 1 0 .67 0, .70 0, .79 Balsam f i r Length 2 0 .30 0. .61 0, .62 Shoot 2 1 0 .72 0, .76 0, .77 Balsam f i r Length 2 0 .49 0, .74 0. .58 Basal 2 1 0 .69 0, .76 0. .76 L o b l o l l y pine Area 2 0 .36 0, .49 0, .56 Height 2 1 0 .81 0. .86 0, .90 L o b l o l l y pine 2 0 .82 0. .87 0. .88 Basal 3 1 0 .66 0. .85 0. .65 Jack pine Area 2 0 .31 0, .36 0, .69 Volume 5 1 0 .59 0. .66 0. .58 Douglas f i r 2 0 .62 0, .81 0. .93 35 d i a g n o s t i c purposes. T h i s has been extended to the use of m u l t i p l e r e g r e s s i o n to i n f e r whether one or more n u t r i e n t s may be l i m i t i n g growth. These r e g r e s s i o n procedures are a p p l i e d to untreated stands. Those elements with the most s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n c o e f f i c i e n t s are considered to be d e f i c i e n t and those with zero and negative c o r r e l a t i o n c o e f f i c i e n t are c o n s i d e r e d to be s u f f i c i e n t and t o x i c , r e s p e c t i v e l y . Regression a n a l y s i s can be problematic i f a l i m i t i n g n u t r i e n t i s i n v a r i a n t , i f the v a r i a b i l i t y of a n o n - n u t r i t i o n a l f a c t o r i s unaccounted f o r , or i f measured v a r i a b l e s l i e o u t s i d e the range where the r e l a t i o n s h i p s are l i n e a r . Regression a n a l y s i s has been used by Leyton and Armson (1955) i n Scots pine, Leyton (1956, 1957) i n Japanese l a r c h , and P r u s i n k i e w i c z (1982) i n Scots pi n e . White and Mead (1971) demonstrated the use of m u l t i v a r i a t e d i s c r i m i n a n t a n a l y s i s of f o l i a r n u t r i e n t s to help d i s t i n g u i s h between t r e e s having green and y e l l o w f o l i a g e . The second stage i n n u t r i e n t d i a g n o s i s i n v o l v e s demonstration of the d e f i c i e n c y or t o x i c i t y . The demonstration of a d e f i c i e n c y or t o x i c i t y r e q u i r e s the a p p l i c a t i o n of the n u t r i e n t s i n q u e s t i o n , through s c r e e n i n g t r i a l s , and measurement of the subsequent n u t r i e n t uptake and growth. If a stand i s i n the d e f i c i e n t zone one would expect to o b t a i n a response to a wide range of n u t r i e n t l e v e l s . Regression ( l i n e a r , m u l t i p l e or c u r v i l i n e a r ) can a l s o be a p p l i e d to analyse or i n t e r p r e t growth response to n u t r i e n t 36 treatments. Regression models have been produced i n which f o l i a r n u t r i e n t c o n c e n t r a t i o n s have been c o r r e l a t e d with some f u n c t i o n of growth such as height, s i t e index, volume p r o d u c t i o n , mean or p e r i o d i c increment (Bevege 1984). I n t e r p r e t a t i o n of n u t r i e n t growth response data using f o l i a r n u t r i e n t c o n c e n t r a t i o n and growth alone can be p r o b l e m a t i c due to the c o n c e n t r a t i o n and d i l u t i o n e f f e c t s . To overcome these problems a second method, v e c t o r a n a l y s i s as d e s c r i b e d by Timmer and Stone (1978), s i m u l t a n e o u s l y examines n u t r i e n t c o n c e n t r a t i o n , n u t r i e n t content and f o l i a r mass. The v e c t o r a n a l y s i s method and i t s i n t e r p r e t a t i o n are presented i n Figure 6 with a d d i t i o n a l i n t e r p r e t a t i o n s by J a r r e l l and B e v e r l y presented i n Table 5. I n t e r p r e t a t i o n of growth response i s based upon the d i r e c t i o n and extent of the s h i f t of the v e c t o r . A t h i r d method, the boundary l i n e model, uses data accumulated through f i e l d surveys to i d e n t i f y optimum f o l i a r n u t r i e n t values ( F i g u r e 7). When a p l a n t i s c l o s e to i t s optimum n u t r i e n t value there o f t e n i s very l i t t l e r e l a t i o n s h i p between n u t r i t i o n and growth (Sumner 1978) which i s represented by the boundary l i n e model. In t h i s s i t u a t i o n other i n t e r a c t i n g f a c t o r s cannot be c o n t r o l l e d with the r e s u l t being the data represented as an a r r a y of p o i n t s . The s c a t t e r of p o i n t s may be due to e r r o r s of measurement, v a r i a b i l i t y of the b i o l o g i c a l m a t e r i a l and the o v e r a l l v a r i a t i o n caused by other i n t e r a c t i n g f a c t o r s (Webb 37 F O L I A R MASS (g s h o o t " 1 ) • ELEMENT CONTENT ( u g s h o o t - 1 ) DIRECTION OF SHIFT RESPONSE IN CHANGE IN NEEDLE NUTRIENT NUTRIENT POSSIBLE WEIGHT CONC. CONTENT STATUS DIAGNOSIS A B C D F + + + O O + + ++ + + + + ± DILUTION UNCHANGED DEFICIENCY LUXURY CONSUMPTION EXCESS EXChSS NON-LIMITING NON-LIMITING LIMITING NON-TOXIC TOXIC ANTAGONISTIC F i g u r e 6 . V e c t o r m e t h o d f o r t h e i n t e r p r e t a t i o n o f n u t r i e n t - g r o w t h r e s p o n s e d a t a u s i n g n u t r i e n t c o n c e n t r a t i o n , n u t r i e n t c o n t e n t and d r y mass o f n e e d l e s ( f r o m l i m n e r and S t o n e 1 9 7 8 ) ) . 3a Table 5. General r e p r e s e n t a t i o n of changes i n t o t a l content, y i e l d and c o n c e n t r a t i o n as a f f e c t e d by imposed treatments. An i n c r e a s e i s represented by ( I ) , a decrease by (D) and no change by (0) (From J a r r e l l and B e v e r l y (1981). Case Change i n Comments Content Y i e l d C o n c e n t r a t i o n I I I I Synergism 2 I 1 0 3 I I D D i l u t i o n 4 1 0 I Synergism 5 I D I C o n c e n t r a t i o n 6 0 0 0 No Response 7 D I D D i l u t i o n 8 D O D Antagonism 9 D D I C o n c e n t r a t i o n 10 D D 0 11 D D D Antagonism 39 0 _| , ' ' * - i 0.00 0.02 0.04 0.06 LEAF N/DM F i g u r e 7 . An e x a m p l e o f a b o u n d a r y l i n e c o n f i n i n g t h e d a t a r e p r e s e n t i n g o v e r 8 , 0 0 0 d a t a p o i n t s o f m a i z e y i e l d v e r s u s l e a f n i t r o g e n : d r y m a t t e r ( N : D M ) ( g k g - 1 ) ( f r o m W a l w o r t h a n d S u m n e r ( 1 9 8 8 ) ) . 40 1972). T h i s method s e t s about c o n s c i o u s l y v a r y i n g the c o n t r o l l a b l e growth f a c t o r s as much as p o s s i b l e by c o l l e c t i n g a bank of o b s e r v a t i o n s that represent the v a r i a b i l i t y encountered i n the r e a l world (Walworth et al. 1986). T h e r e f o r e , the data a c t u a l l y r epresent d i f f e r e n t response curves from s i n g l e - f a c t o r experiments as i n F i g u r e 8. A response curve from a s i n g l e - f a c t o r experiement may f o l l o w any one of the curves depending on the degree of y i e l d l i m i t a t i o n exerted by other f a c t o r s . Curve f i t t i n g may be used to f i t a model which d e s c r i b e s the boundary l i n e response s u r f a c e . A p o i n t on the boundary l i n e r e p r e s e n t s the maximum a t t a i n a b l e y i e l d at a given f o l i a r c o n c e n t r a t i o n under a given s e t of c o n d i t i o n s (Walworth and Sumner 1988). T h i s i s not the same as the maximum y i e l d a t t a i n a b l e where a l l growth f a c t o r s are optimal (Sumner 1978). i i . T o t a l A n a l y s i s using N u t r i e n t Balances A number of other methods take n u t r i e n t balance i n t o account. The nutrient-element balance concept was introduced by Shear er al. (1946). Sumner (1978) used the boundary l i n e model with n u t r i e n t r a t i o s . He then c o n s i d e r e d a number of r a t i o s s i m u l t a n e o u s l y . By combining the i n f o r m a t i o n obtained from each r a t i o , the order i n which the p l a n t r e q u i r e s these n u t r i e n t s i s o b t a i n e d . Prevot's f a c t o r i a l method uses f a c t o r i a l experiments to 41 Zone of Balanced Zone of X Insufficiency Nutrition X Excess or or Y Excess Y Insufficiency Soil Nutrient (X) Level • or Tissue Nutrient Ratio (X/Y) F i g u r e 8 . D i a g r a m m a t i c r e p r e s e n t a t i o n o f c r o p r e s p o n s e t o a number o f l i m i t i n g f a c t o r s ( f r o m W a l w o r t h and S u m n e r ( 1 9 8 8 ) ) . 42 study and c a l i b r a t e i n c r e a s i n g l e v e l s of one or more f a c t o r s while a l l other c o n d i t i o n s are kept c o n s t a n t . From these c a l i b r a t i o n s one i s able to determine the r e l a t i v e p r o p o r t i o n s of i n t e r a c t i n g n u t r i e n t s which would r e s u l t i n balanced n u t r i t i o n . Kenworthy's balance i n d i c e s (Walworth and Sumner 1988) are based on f o l i a r optima generated by averaging t i s s u e values of h e a l t h y p l a n t s gathered from survey data. These values are s p e c i f i c to the stage of growth and p o s i t i o n of the sampled f o l i a g e . The r e s u l t s from a n a l y s i s of a sample are compared to the norms and a l s o weighted using c o e f f i c i e n t s of v a r i a t i o n which represent the normal v a r i a t i o n s of the standard v a l u e s . T h i s i s done using n u t r i e n t i n d i c e s which are c a l c u l a t e d as f o l l o w s . Balance Index = ( x / s HlOO + ( i - ( x / s ) ) ( c v ) when x < s Balance Index « (x/s)(100 - (1 - ( x / s ) ) ( c v ) when x > s where x = n u t r i e n t c o n c e n t r a t i o n s » standard (optimum) value cv = c o e f f i c i e n t of v a r i a t i o n ( i n percent) of s N u t r i e n t s are then ranked i n order of requirement with the n u t r i e n t having the lowest index being the most r e q u i r e d . The N o l l e r - N i e l s e n Technique attempts to overcome problems of p h y s i o l o g i c a l age and n u t r i e n t i n t e r a c t i o n s . Four steps are 43 i n v o l v e d . The f i r s t step i s the pr o d u c t i o n of response curves from n u t r i e n t response experiments. With the a i d of these curves i n d i v i d u a l p l a n t samples are c o r r e c t e d back to a standard p l a n t mass. In the second step, standard n u t r i e n t values are developed from f a c t o r i a l experiments using the boundary l i n e method to determine optimal c o n c e n t r a t i o n s . The p l a n t samples are then compared to the boundary l i n e values and the maximum a t t a i n a b l e y i e l d i s determined. The most l i m i t i n g n u t r i e n t i s the one with the lowest maximum y i e l d . In the t h i r d s t ep, boundary l i n e curves f o r p l o t s of i n t e r a c t i n g n u t r i e n t s are used to determine the optimum l e v e l s of other n u t r i e n t s at the e x i s t i n g l e v e l of the m o s t - l i m i t i n g n u t r i e n t . The f i n a l p r e d i c t e d y i e l d i s determined by e s t i m a t i n g the y i e l d r e d u c t i o n due to each n u t r i e n t . The DRIS method ( D i a g n o s t i c and Recommendation Integrated System) (Walworth and Sumner 1988) attempts to overcome problems a s s o c i a t e d with p l a n t age, n u t r i e n t i n t e r a c t i o n s , and f o l i a r optima d e t e r m i n a t i o n . F o l i a r optima are c a l c u l a t e d by averaging n u t r i e n t l e v e l s from h e a l t h y or high y i e l d i n g p l a n t s . The d e v i a t i o n s from the mean optimum value are estimated by the c o e f f i c i e n t s of v a r i a t i o n of the high y i e l d i n g p l a n t s . I n d i c e s are then c a l c u l a t e d f o r each n u t r i e n t using n u t r i e n t r a t i o s i n the f o l l o w i n g e q u a t i o n s . A index • (f(A/B) + f(A/C) + f(A/D) .+ f ( A / M ) ) / 2 44 where f(A/B) - ( ( ( A / B ) / ( a / b ) ) - l ) ( 1 0 0 0 / c v ) when (A/B) > (a/b) where f(A/B) = ( l - ( ( a / b ) / ( A / B ) ) ) ( t O O O / c v ) when (A/B) < (a/b) A/B i s the value of the r a t i o of the two elements i n the t i s s u e under d i a g n o s i s a/b i s the value of the corresponding norm z i s the number of f u n c t i o n s cv i s the c o e f f i c i e n t of v a r i a t i o n of the norm The magnitude of each n u t r i e n t index r e p r e s e n t s the r e l a t i v e excess ( p o s i t i v e value) or d e f i c i e n c y (negative value) of the n u t r i e n t i n the t i s s u e . Ingestad (1979) developed a method using n u t r i e n t balance. Nitrogen i s expressed as 100 and a l l other n u t r i e n t s are expressed r e l a t i v e to n i t r o g e n . A set of r a t i o s was developed f o r macronutrients i n hemlock, and f o r m i c r o n u t r i e n t s i n some c o n i f e r s of the f a m i l y Pinaceae (Ingestad 1979). i i i . A n a l y s i s of Separate F r a c t i o n s F o l i a r c o n c e n t r a t i o n s i n themselves do not n e c e s s a r i l y i n d i c a t e anything about the p h y s i o l o g i c a l requirements of a n u t r i e n t . With w i l d p l a n t s , n u t r i e n t l e v e l s may serve both a 45 p h y s i o l o g i c a l as well as an e c o l o g i c a l f u n c t i o n i n the p l a n t ' s s t r a t e g y , as i n accumulator p l a n t s . T o t a l l e v e l s may not n e c e s s a r i l y be r e f l e c t i v e of the p h y s i o l o g i c a l n u t r i t i o n of the p l a n t because of compartmentation of n u t r i e n t s or d i f f e r e n c e s i n the d i s t r i b u t i o n of n u t r i e n t s among chemical f r a c t i o n s . T h e r e f o r e , i t would be d e s i r a b l e to measure the p h y s i o l o g i c a l l y a c t i v e f r a c t i o n of a n u t r i e n t i n order to diagnose the n u t r i e n t s t a t u s of a p l a n t and c o r r e l a t e that n u t r i e n t c o n c e n t r a t i o n with bio c h e m i c a l and p h y s i o l o g i c a l a c t i v i t i e s . The " f u n c t i o n a l n u t r i e n t requirement" of a p l a n t i s "the minimal n u t r i e n t c o n c e n t r a t i o n which can s u s t a i n i t s metabolic f u n c t i o n at r a t e s which do not l i m i t growth" (Ohki 1987). These methods i n v o l v e i s o l a t i o n and a n a l y s i s of separate c e l l u l a r components, a n a l y s i s of d i f f e r e n t chemical f r a c t i o n s , enzyme a n a l y s i s , the measurement of p h y s i o l o g i c a l processes, the use of e x t r a c t a n t s , in situ a n a l y s i s , and the use of c e l l c u l t u r e s . The methods which have been used f o r Zn and Mn w i l l be d i s c u s s e d . Memon and Yatazawa (1984) i s o l a t e d d i f f e r e n t c e l l u l a r components of Mn accumulator p l a n t s using d i f f e r e n t i a l c e n t r i f u g a t i o n . The c e l l w a l l m a t e r i a l , c h l o r o p l a s t s , mitochondria, ribosomes, and vacuolar contents were separated using t h i s method. 46 Enzyme a c t i v i t y has been used as a method to diagnose n u t r i e n t s t a t u s . The a c t i v i t y of the Mn isozyme of superoxide dismutase (Mn-SOD) i n pea leaves was d i r e c t l y r e l a t e d to the Mn n u t r i e n t l e v e l s ( d e l Rio et al. 1978). I t was suggested that t h i s isozyme could be an i n d i c a t o r of the b i o l o g i c a l l y a c t i v e Mn i n v o l v e d i n c e l l metabolism ( d e l Rio er al. 1978). The a c t i v i t y of IAA oxidase was found to i n c r e a s e i n c o t t o n with Mn t o x i c i t y (Morgan er al. 1966). In bean l e a v e s , Mn t o x i c i t y i n c r e a s e d the a c t i v i t i e s of i s o c i t r i c dehydrogenase and malic enzyme (Anderson and Evans 1956). The a c t i v i t y of r i b o n u c l e a s e (RHAase) was found to be a s e n s i t i v e , r e l i a b l e and b e t t e r index f o r d e t e c t i n g Zn d e f i c i e n c y i n r i c e and maize than Zn c o n c e n t r a t i o n (Dwivedi and Takkar 1974). There was an i n v e r s e r e l a t i o n s h i p between Zn supply and RNAase a c t i v i t y . Carbonic anhydrase a c t i v i t y i s r e l a t e d to the Zn n u t r i e n t l e v e l . A shortage i n Zn supply to spinach d r a s t i c a l l y reduced c a r b o n i c anhydrase l e v e l s (Randall and Bouma 1973). Carbonic anhydrase a c t i v i t y from f o l i a g e was used to d e t e c t Zn d e f i c i e n c i e s i n pecan ( S n i r 1983), and i n maize (Gibson and Leece 1981). Rahimi and Schropp (1984) a l s o used c a r b o n i c anhydrase a c t i v i t y as an i n d i c a t i o n of the Zn n u t r i t i o n a l s t a t u s of maize, m i l l e t , tobacco, sugar beet and grape. The a c t i v i t y of SOD which was a Cu-Zn isozyme i n c o t t o n l e a v e s was used as an i n d i c a t o r of Zn s t a t u s (Cakmak and Marschner 1987). Water-soluble Zn from f o l i a g e has been found to be a s u i t a b l e i n d i c a t o r of Zn n u t r i t i o n a l s t a t u s . Cakmak and 47 Marschner (1987) found the c o n c e n t r a t i o n of water-soluble Zn i n lea v e s to be c l o s e l y c o r r e l a t e d with v i s u a l Zn d e f i c i e n c y symptoms and superoxide dismutase i n c o t t o n . In orange t r e e s , v i s u a l Zn d e f i c i e n c y symptoms i n lea v e s were c l o s e l y r e l a t e d to the c o n c e n t r a t i o n of water-soluble Zn (Cakmak and Marschner 1987). T h i s supports the work of Rahimi and Schropp (1984) who al s o found that water-soluble Zn was a b e t t e r i n d i c a t o r of Zn n u t r i t i o n a l s t a t u s than was t o t a l Zn. Th i s was due to the d i r e c t r e l a t i o n s h i p between water-soluble Zn and the a c t i v i t y of ca r b o n i c anhydrase. The p o s s i b i l i t y of q u a n t i f y i n g the l e v e l s of n u t r i e n t s i n p l a n t s i n situ and examining t h e i r d i s t r i b u t i o n has been demonstrated using X-ray m i c r o a n a l y s i s . The f i r s t system which has been used i s e l e c t r o n probe X-ray m i c r o a n a l y s i s (EPMA). In t h i s system, e l e c t r o n s e x c i t e atoms i n a sample, r e s u l t i n g i n the pro d u c t i o n of c h a r a c t e r i s t i c X-ray p a t t e r n s which can be measured. A scanning e l e c t r o n microscope i s used i n c o n j u n c t i o n with the e l e c t r o n microprobe. A d e s c r i p t i o n of the instrument and i t s a p p l i c a t i o n i n b i o l o g y has been given by H a l l (1979). EPMA has been used by Memon et al. (1980) and Memon and Yatazawa (1982) l o o k i n g at Mn i n Mn-accumulators, by Ho r i g u c h i and Mo r i t a (1987) who looked at Mn i n leaves of b a r l e y , and f o r the study of Zn accumulation i n the r o o t s of Betula (Denny and W i l k i n s (1987), and i n the ro o t s of Deschampsia caespitosa (Van Steveninck ec al. 1987). 48 A second instrument which has begun to be used i n p l a n t r e s e a r c h f o r in situ a n a l / s i s i s the scanning proton mlcroprobe (SPM). A d e s c r i p t i o n of the instrument and i t s use i n b i o l o g i c a l r e s e a r c h has been reviewed by Legge and N a z z o l i n i (1980), Legge (1982), Enderer (1982), Legge e t al. (1982), Legge (1980), and Legge er al. (1979). T h i s combines a scanning mode with a proton mlcroprobe which u t i l i z e s proton-induced X-ray emission (PIXE) (Legge er al. 1979). SPM has enhanced s e n s i t i v i t y compared to EPMA-SEM because the background r a d i a t i o n which i s produced (Bremsstrahlung) i s orders of magnitude lower ( M a z z o l i n i er al. 1985). Consequently, t h i s allows one to de t e c t elements i n a sample down to 1 pg g~* (Legge et al. 1979). The SPM instrument could a l s o be used to de t e c t and measure i s o t o p e s using nuclear s c a t t e r i n g (Legge er al. 1979). The a p p l i c a t i o n of the PIXE i n pla n t n u t r i t i o n s t u d i e s has been demonstrated by M a z z o l i n i er al. (1985) using wheat seeds, and i n the f o l i a g e of Eucalyptus obliqua ( M a z z o l i n i er al. 1982). The instrument allows one to produce a q u a n t i t a t i v e elemental a n a l y s i s of d i f f e r e n t t i s s u e s , as well as producing elemental maps showing the d i s t r i b u t i o n of the elements i n the t i s s u e . G. Some Con c l u s i o n s Hemlock i s a s p e c i e s which may be c l a s s i f i e d as a c a l c i f u g e p l a n t because hemlock has i n common with t h i s group the f o l l o w i n g c h a r a c t e r i s t i c s . These p l a n t s favor a c i d s o i l s and they have ammonium-based n i t r o g e n n u t r i t i o n . P l a n t s which 49 u t i l i z e ammonium-nitrogen can i n f a c t promote a c i d i f i c a t i o n of the surrounding s o i l . A c i d s o i l c h a r a c t e r i s t i c a l l y has high p l a n t a v a i l a b l e l e v e l s of aluminium and manganese. Hemlock t h r i v e s under these a c i d s o i l c o n d i t i o n s . Hemlock a l s o f a v o r s o r g a n i c r i c h media which may keep Mn i n the reduced s t a t e . T i s s u e n u t r i e n t c o n c e n t r a t i o n s i n a p l a n t are not n e c e s s a r i l y r e f l e c t i v e of the p h y s i o l o g i c a l requirements f o r that p a r t i c u l a r n u t r i e n t . T h i s i s evident f o r Mn where there i s abundant evidence that Mn l e v e l s i n p l a n t s are not r e f l e c t i v e of p h y s i o l o g i c a l requirements. N u t r i e n t s not onl y have a p h y s i o l o g i c a l r o l e but may a l s o p l a y a r o l e i n the e c o l o g i c a l s t r a t e g y of a p l a n t . There are three types of e c o l o g i c a l s t r a t e g i e s of p l a n t n u t r i t i o n : the accumulator, the excluder and the i n d i c a t o r . Western hemlock has lower f o l i a r Zn l e v e l s and higher f o l i a r Mn l e v e l s as compared to c o n i f e r s i n other genera i n B.C. and the U.S. P a c i f i c Northwest. Whether these d i f f e r e n c e s r epresent a c t u a l d i f f e r e n t p h y s i o l o g i c a l requirements of s p e c i e s , or are an i n d i c a t i o n of a Zn d e f i c i e n c y and Mn t o x i c i t y i n hemlock r e q u i r e s some i n v e s t i g a t i o n , due to the p o s s i b i l i t y of i n c r e a s i n g p r o d u c t i v i t y through a l l e v i a t i o n of these s t r e s s e s . A review of the l i t e r a t u r e leads to some c o n c l u s i o n s u s e f u l i n s e l e c t i n g r e s e a r c h methods. The c l a s s i c a l method (a s i n g l e a p p l i c a t i o n of a f e r t i l i z e r dosage) was used due to the 50 g r e a t e r ease i n e s t a b l i s h i n g and c a r r y i n g out the treatments. D i a g n o s i s of f o l i a r data was performed using the vecto r method. I n v e s t i g a t i o n of the p h y s i o l o g i c a l a c t i v e f r a c t i o n s of Zn and Mn were made using analyses of e x t r a c t i o n s and of f o l i a r c e l l u l a r f r a c t i o n s . 51 CHAPTER 3. METHODS AMD MATERIALS A. S i t e D e s c r i p t i o n 1. L o c a t i o n of Study Areas F e r t i l i z e r s c r e e n i n g t r i a l s were e s t a b l i s h e d i n f i v e d i f f e r e n t stands i n the Vancouver F o r e s t Region i n C o a s t a l B r i t i s h Columbia ( F i g u r e 9 ) . Information on the l o c a t i o n of the stands i s given i n Appendix A. Three of the stands are l o c a t e d at the U n i v e r s i t y of B r i t i s h Columbia Research F o r e s t , r e f e r r e d to as s i t e s 1, 2 and 4. The remaining two stands are l o c a t e d at Chipmunk Creek ( C h i l l i w a c k P r o v i n c i a l F o r e s t ) and M i s s i o n Tree Farm r e f e r r e d to as s i t e s 3 and 5 r e s p e c t i v e l y . S i t e s were s e l e c t e d on the b a s i s of t h e i r p r o x i m i t y to Vancouver, s u p p o r t i n g a uniform stand of t r e e s and c o n s i s t i n g of enough t r e e s to support a f e r t i l i z e r t r i a l . S i t e s were not s e l e c t e d on the b a s i s of any s p e c i f i c s i t e c h a r a c t e r i s t i c . , S i t e s were c l a s s i f i e d a c c o r d i n g to K l i n k a er al. (1984). S i t e 1 i s l o c a t e d i n the P a c i f i c Ranges D r i e r Maritime C o a s t a l Western Hemlock b i o g e o c l i m a t i c subzone. According to K l i n k a et al. (1984), the c l i m a t e i s c h a r a c t e r i z e d by 57 mm of p r e c i p i t a t i o n i n the d r i e s t month, warm summers (17.6°C warmest month mean, 33.3°C absolute maximum), mild w i n t e r s , no month with a mean minimum temperature below 0°C, a mean annual p r e c i p i t a t i o n 52 53 of 1860 no, with 5% as snow, and a range between minimum winter and maximum summer means of 15.2°C. S i t e s 2, 3, 4, and 5 are l o c a t e d i n the Windward Montane Maritime Wetter C o a s t a l Western Hemlock b i o g e o c l i m a t i c subzone. According to K l i n k a e t al. (1984), the c h a r a c t e r i s t i c c l i m a t e i n t h i s zone i s i n t e r m e d i a t e between the Windward Submontane Maritime C o a s t a l Western Hemlock zone and the Maritime Forested Mountain Hemlock zone. A c l i m a t e s t a t i o n i s not l o c a t e d i n t h i s v a r i a n t ; t h e r e f o r e there are no c l i m a t e data. 2. Stand C h a r a c t e r i s t i c s S i t e s 1, 2, 3, 4, and 5 were composed of young uneven-aged western hemlock of n a t u r a l o r i g i n . In 1987, the estimated age of the t r e e s ranged from about 10 to 20 years o l d and they were "free-growing". These stands were c h a r a c t e r i s t i c of western hemlock, having clumps of t r e e s with the spaces between clumps being f i l l e d i n by i n d i v i d u a l t r e e s . Crown c l o s u r e had not yet occurred i n these stands. F u r t h e r d e s c r i p t i o n s on the s p e c i e s composition of these stands i s presented i n Appendix A. 3. S o i l C h a r a c t e r i s t i c s S o i l p r o f i l e s of the f i v e s i t e s were d e s c r i b e d a c c o r d i n g to the Canadian system of s o i l c l a s s i f i c a t i o n (Canada S o i l Survey Committee 1978). The s o i l s at s i t e s 1 and 3 were c l a s s i f i e d as O r t h i c Humo-Ferric Podzols, at s i t e 2 as a Rego G l e y s o l , at s i t e 54 4 as a Duric Humo-Ferric Podzol, and at s i t e 5 as an O r t h i c Ferro-Humic Pod z o l . The chemical c h a r a c t e r i s t i c s of the s o i l p r o f i l e s are presented i n Table 6 and other s o i l c h a r a c t e r i s t i c s i n Appendix A. The methods used f o r chemical a n a l y s i s of the s o i l s are d e s c r i b e d i n s e c t i o n G. B. Experimental Design The experiment was c a r r i e d out using s i n g l e t r e e s as p l o t s ( V i r o 1967) i n a randomized block design with the blocks a c t i n g as r e p l i c a t e s . There were ten r e p l i c a t e s per treatment at each of the s i t e s . The number, types and year of treatments v a r i e d between s i t e s (Table 7 ) . On each s i t e , t r e e s were s e l e c t e d a c c o r d i n g to the g u i d e l i n e s given by B a l l a r d and C a r t e r (1986). Dominant and codominant t r e e s were s e l e c t e d which were devoid of d e f o r m i t i e s , i n s e c t and d i s e a s e s , and cone c r o p s . The minimum d i s t a n c e between s e l e c t e d t r e e s was s i x meters. There was a compromise between having t r e e s c l o s e enough to decrease the e f f e c t of s i t e v a r i a b i l i t y , but f a r enough to prevent contamination from adjacent treatments. The t r e e s were i d e n t i f i e d with p l a s t i c t a g s . T a b l e 6. C h e m i c a l c h a r a c t e r i s t i c s o f t h e s o i l p r o f i l e s . SITE HORIZON DEPTH Zn Hn K Ca Mg P Al -cm — 1 1 LFH 3-0 11.8 67 .2 221 718 57 .0 33 1891 1 2 Bf 0-42 1.4 7 . 4 43 62 6 .8 0 2084 1 3 C 42-52 0.9 2 .0 12 48 7 .3 0 2148 2 1 LFH 15-0 67.0 72 .0 295 2566 528 . 0 131 814 2 2 Cg 0-18 7.8 5 .7 129 619 77 .0 52 991 3 1 L(f ) 2-0 2 . 1 2 . 1 74 115 35 .0 0 2052 3 2 Ae 0-5 1.1 9 .3 105 65 16 .0 0 2114 3 3 Bf 5-20 0.5 5 .0 255 16 3 . 0 0 2102 3 4 Bf 20-35 0.3 3 .0 51 8 1 .4 0 2099 3 5 Bf 35-60 0.3 3 .0 0 6 1, .0 0 1894 3 6 Cg 60 + 0.5 6 .0 23 5 0 . 8 43 1710 4 L LFH 20-0 39 .0 129 .0 404 1782 224. .0 51 1049 4 2 Ae 0-10 0.8 3 .2 37 96 3 . ,0 0 2022 4 3 Bf 10-46 1.9 9 .7 0 26 5 . ,6 0 2079 4 4 BCc 46-72 2.2 20 .0 0 47 6 . ,4 2 2039 4 5 C 72 + 0.5 0 .5 48 24 2 . ,2 0 2075 5 1 LFH 25-0 30.3 51 .7 212 2471 234. 0 26 1412 5 2 Bhf 3-6 3.6 2 . 4 86 369 52 . 0 6 1869 5 3 Bhf 6-18 0.6 0 .4 0 45 8 . 3 0 2123 5 4 Bf 18-27 0.7 0 .5 45 53 10 . 0 0 2141 5 5 Bf 27-35 1.1 1 .2 78 80 17. 0 0 2054 5 6 C 35 + 0.3 0 . 4 57 25 2. 3 0 2123 SITE HORIZON DEPTH Fe Cu B Extr . N Total CEC PH g" 1 - 1 _ meq 100 g" 1 HaO 1 1 LFH 3-0 249 1. 5 0 0.006 0.062 6 . 0 3 .9 t 2 Bf 0-42 48 1. 1 0 0.003 0.195 12 . 1 4 .9 1 3 C 42-52 204 0. 9 0 0.005 0.213 21, .7 4 .5 2 1 LFH 15-0 174 1. 6 0 0.015 0.907 16 . 0 3 .6 2 2 Cg 0-18 157 1. 0 0 0.003 0.218 14, .0 3 .7 3 1 L(f ) 2-0 381 0 . 4 0.2 0.003 0.076 20 , .3 3 .8 .8 3 2 Ae 0-5 106 1. ,0 0 .08 0.006 0.039 12 .5 4 3 3 Bf 5-20 106 0. 7 0 .06 0.003 0.076 9 . 9 4 .8 3 4 Bf 20-35 51 0 . 7 0.05 0.002 0.057 4, . 7 4 .9 3 5 Bf 35-60 71 0.5 0.04 0.003 0.028 1, .0 4 . 8 3 6 Cg 60 + 40 0 . ,4 0.03 0.004 0.016 0 4 .8 4 1 LFH 20-0 238 2 . 2 0 0.016 0.709 12 , . 1 3 .6 4 2 Ae 0-10 59 1. .1 0 0.002 0.076 5 , .1 5 .0 4 3 Bf 10-46 125 1. 1 0.08 0.002 0.04 0 . 8 4 . 7 4 4 BCc 46-72 130 0 . 8 0.07 0.004 0.256 9 . 5 4 .4 4 5 C 72 + 68 1. 0 0.03. 0.005 0.072 1. .9 4 .4 5 1 LFH 25-0 223 2 . 2 0 0.008 0.755 6 , .9 4 .2 5 2 Bhf 3-6 135 2 . ,2 0 .08 0.007 0.074 24, .0 4 . 3 5 3 Bhf 6-18 44 0. .8 0 .03 0.003 0.233 21, .2 4 4 . 6 5 4 Bf 18-2 7 67 1. .0 0.02 0.005 0.317 13 , .0 .6 5 S Bf 27-35 88 1. .0 0 .001 0.005 0.259 15 , .1 4 .5 5 6 C 35 + 20 1. .2 0 . 1 0.001 0.09 5 , .6 4 . 9 pH 3.4 4.4 4.1 3.2 3.1 56 Table 7. Tr e a t m e n t - l e v e l s used i n the f e r t i l i z e r t r i a l s . S i t e s 1, 2, and 3 A p p l i c a t i o n Method S o i l Number 1 2 3 9 10 12 ZnSO* ZnSO* MnSO* NaaSO* NaaSO* C o n t r o l Rate 10 kg Zn h a " 1 50 kg Zn h a - 1 200 kg Mn h a ~ v 5 kg S ha~* 25 kg S ha"* F o l i a r 4 5 6 7 8 t l ZnSO* ZnSO* MnSO* HaaSO*. NaaSO A C o n t r o l 360 mg Zn L~x 3600 mg Zn L~x 2730 mg Mn L ~ x 177 mg S L - 1 1770 mg S L ~ x Demin HaO + S u r f a c t a n t Table 7 (continued) S i t e s 4 and 5 S o i l 1 2 3 4 5 6 7 8 9 10 11 12 F o l i a r 13 14 15 16 17 18 19 20 21 22 24 ZnSO* 50 kg Zn ha~ X ZnSO* 100 kg Zn ha~ 1. ZnSO* 200 kg Zn ha~ X MnSOd. 200 kg Mn ha~ X MnS0 A 400 kg Mn ha~ X MnSO* 600 kg Mn ha~ X S 25 kg S ha~* S 49 kg S h a " 1 S 98 kg S h a " 1 s 118 kg S h a " 1 s 235 kg S h a " 4 s 352 kg S ha~* ZnSO* 360 mg Zn L~* ZnSO* 1800 mg Zn L~* ZnSO^ 2700 mg Zn L _ 1 MnSO* 2730 mg Mn L ~ l MDSOA 4095 mg Mn L~* NaaSO* 177 mg S L~x NaaSO* 883 mg S L-«- HaaSO A 1325 mg S NaaSO A 1605 mg S L~x NaaSOA 2408 mg S L " 1 C o n t r o l Demin HaO + S u r f a c t a n t Table 7 (concluded) S o i l 23 "Complete -Zn -Mn" Urea 100 kg N h a _ t T r i p l e Super Phosphate 150 kg P h a " 1 K S S O A 22 kg K h a " 1 MgSO* 18 kg Mg h a " 1 CaSO* 11 kg Ca h a " 1 Degra-sul 22 kg S h a " 1 CUSOA 4.4 kg Cu ha~ Solubor 0.9 kg B h a " 1 FeSO A 11 kg Fe h a " 1 25 C o n t r o l 59 C. F e r t i l i z e r Treatments F e r t i l i z a t i o n can be used as a t o o l to diagnose the n u t r i e n t s t a t u s of a stand. The purpose i s to i n c r e a s e the supply of the n u t r i e n t of i n t e r e s t . Then, using the dose- response curve and vector a n a l y s i s , one can evaluate the n u t r i e n t s t a t u s of the stand. The f e r t i l i z e r s used were z i n c s u l p h a t e , manganese su l p h a t e , sodium s u l p h a t e , elemental sulphur (as Degra-sul), urea, t r i p l e super phosphate, potassium s u l p h a t e , c a l c i u m s u l p h a t e , magnesium s u l p h a t e , i r o n s u l p h a t e , copper sul p h a t e , and boron (as S o l u b o r ) . The s p e c i f i c treatments used f o r each s i t e are o u t l i n e d i n Table 7. There were a t o t a l of 120 t r e e s i n the t r i a l s on each of s i t e s 1, 2, and 3, and 250 t r e e s on each of s i t e s 4 and 5. F e r t i l i z e r was a p p l i e d i n s o l i d form to the s o i l and i n s o l u t i o n as a spray to the f o l i a g e . The s o i l and f o l i a r treatments were a p p l i e d at d i f f e r e n t times. The s o i l treatments were a p p l i e d i n May, and the f o l i a r treatments were a p p l i e d at the beginning of J u l y when there was a l a r g e amount of new growth. For s i t e s 1, 2 and 3, the t r i a l s were e s t a b l i s h e d i n 1985. For s i t e s 4, and 5 the t r i a l s were e s t a b l i s h e d i n 1986. On s i t e s 1, 2, and 3, sodium sulphate was used as the sulphur source f o r the f o l i a r and s o i l treatments. On s i t e s 4 and 5, elemental sulphur was used as the sulphur source f o r the s o i l treatments 60 and sodium sulphate f o r the f o l i a r treatments. The "complete - Zn-Mn" treatment was a p p l i e d only to s o i l s , and only at s i t e s 4 and 5 . The f e r t i l i z e r s o l u t i o n s were prepared using deraineralized water. A commercial detergent (trade name 'Joy') at a c o n c e n t r a t i o n of 0.5% by volume was added to the f e r t i l i z e r s o l u t i o n as a s u r f a c t a n t to enhance n u t r i e n t a b s o r p t i o n by the f o l i a g e . The f e r t i l i z e r s o l u t i o n s were a p p l i e d using a backpack p l a s t i c s p r a y e r . The t r e e s were sprayed u n t i l the s o l u t i o n began to d r i p from the canopy (approximately one l i t r e per t r e e ) . In t h i s way, i f there were canopy s i z e d i f f e r e n c e s between t r e e s , each u n i t area of f o l i a g e of each t r e e w i l l have had the same ra t e of n u t r i e n t a p p l i c a t i o n . D. F i e l d Sampling The sampling procedure was c a r r i e d out according to the g u i d e l i n e s given by B a l l a r d and C a r t e r (1986). F o l i a g e and shoot samples were c o l l e c t e d from September 15-December 15 from s i t e s 1 and 2 i n 1985, from s i t e s 1, 2, 3, 4, and 5 i n 1986, and from s i t e s 4 and 5 i n 1987. For s i t e s 4 and 5 only r e p l i c a t e s 1-5 were sampled i n 1986. A l l r e p l i c a t e s were sampled i n 1987. For a l l s i t e s and sampling years both the c u r r e n t and pre v i o u s year's f o l i a g e and shoot samples were c o l l e c t e d using hand and pole pruners. F o l i a g e was c o l l e c t e d from the upper one-half to one- quar t e r of the crown but below the t h i r d whorl. A shoot sample 61 r e f e r s to the t i p growth of a branch formed i n the c u r r e n t year. Three shoot samples per t r e e were taken from the t h i r d whorl f o r growth a n a l y s i s . For s i t e 3, shoot samples were taken f o r the c u r r e n t year (1986) and the pre v i o u s two y e a r s . For s i t e s 4 and 5 i n 1987, branch samples were c o l l e c t e d from the t h i r d and f o u r t h whorls from the top c o n s i s t i n g of the pre v i o u s three years of growth f o r r e p l i c a t e s 6-10. This allowed f o r a n a l y s i s of shoot increment f o r 1986 at whorl three f o r a l l 10 r e p l i c a t e s . F o l i a g e and branch samples were placed i n p l a s t i c bags, t r a n s p o r t e d to the l a b o r a t o r y , and s t o r e d i n a c o l d room u n t i l f u r t h e r p r o c e s s i n g . Height increment measurements were taken i n the f a l l of 1986 on s i t e s 1, 2, and 3 f o r the p r e v i o u s three years of growth, and i n 1987 on s i t e s 4 and 5 a l s o f o r the prev i o u s three years of growth. Height increment measurements were made to the nearest 0.5 cm. At the time of sampling t r e e s of other s p e c i e s were a l s o sampled. One codominant or dominant tre e of other s p e c i e s o c c u r r i n g adjacent to the c o n t r o l hemlock tr e e per block was sampled. S o i l samples were c o l l e c t e d from s i t e s 1, 3, 4, and 5 i n the f a l l of the year i n which the f e r t i l i z e r t r i a l was e s t a b l i s h e d . Samples were c o l l e c t e d from around each c o n t r o l hemlock t r e e at a d i s t a n c e of 30 cm from the base of the stem. Up to three samples were c o l l e c t e d at i n t e r v a l s of 120° both of the f o r e s t f l o o r and of the mineral s o i l down to 15 cm. These are i d e n t i f i e d as (T) f o r f o r e s t f l o o r and (B) f o r mineral s o i l . M i n e r a l s o i l s were composited i n the f i e l d . 62 A s o i l p i t was excavated at each of the s i t e s f o r a d e s c r i p t i o n of the p r o f i l e s . In a d d i t i o n , s o i l samples were c o l l e c t e d from H H r . h h o r i z o n f o r o h H m i . c a l . a n a l / H i s . Data from the s o i l p i t were used only f o r d e s c r i p t i v e purposes; no s t a t i s t i c a l i n t e r p r e t a t i o n s were made from the s o i l p i t data. F o l i a g e samples were separated i n t o c u r r e n t and p r e v i o u s - year's p o r t i o n s . Shoot lengths of the c u r r e n t and the previous year's growth were measured to the nearest mm. F o l i a g e was oven- d r i e d at 70°C f o r 24 hours i n paper bags. Growth measurements were made on the samples used f o r the shoot increment measurements. These were f o l i a r mass per shoot to the nearest m i l l i g r a m , and number of needles per shoot. From these values the mass per needle, the needle mass per cm of shoot and the number of needles per cm of shoot were c a l c u l a t e d . In a d d i t i o n , f o r the samples from the c o n t r o l western hemlock t r e e s and samples of t r e e s of other s p e c i e s , the mass of needles per 100 needles was a l s o determined to the nearest m i l l i g r a m . Dry f o l i a g e samples to be used f o r chemical a n a l y s i s were ground i n a Braun type KSM-2 c o f f e e g r i n d e r and s t o r e d i n a i r - t i g h t p l a s t i c c o n t a i n e r s . The shoot samples gave a data base of 2,580 samples f o r both f i r s t and second year. Approximately 490 samples were used to measure f i r s t year n u t r i e n t response, and 860 i n the second 63 year. There were approximately 860 height increment values f o r each of three years f o r the e n t i r e experiment. E. Chemical A n a l y s i s The wet d i g e s t i o n method of Parkinson and A l l e n (1975), s l i g h t l y modified by B a l l a r d (1981) was used f o r the d e t e r m i n a t i o n of t o t a l N, P, K, Ca, Mg, Mn, Zn, and A l ( B a l l a r d and C a r t e r 1986). N and P were measured on the o r i g i n a l d i g e s t by c o l o r i m e t r i c a n a l y s i s f o r N ( p h e n o l - h y p o c h l o r i t e method) and P (unreduced vanadomolybdate complex), by the Technicon Autoanalyzer I I . The elements K, Ca, Mg, Mn, Zn, and A l were measured using atomic a b s o r p t i o n spectrophotometry (AA) ( P e r k i n Elmer 306). For f o l i a g e from 1986 and 1987, the elements P, K, Ca, Mg, Mn, Zn, and.Al were measured using I n d u c t i v e l y Coupled Plasma Emission Spectroscopy (ICP) ( J a r r e l l Ash AtomComp S e r i e s 1100) on the o r i g i n a l d i g e s t s . D e t a i l s of the method are d e s c r i b e d i n Appendix B l . The elements Fe and Cu were determined using a n i t r i c a c i d d i g e s t i o n followed by atomic a b s o r p t i o n spectrophotometry ( B a l l a r d and C a r t e r 1986) (Appendix B2). Although there was a good agreement between the standard f o l i a g e samples using AA and ICP f o r Fe a n a l y s i s using the Parkinson and A l l e n d i g e s t , there was poor agreement for the hemlock samples (Appendix C). Copper was found to be below the d e t e c t i o n l i m i t s of the ICP using the Parkinson and A l l e n d i g e s t s . The standards were used to c o n s t r u c t equations i n order to convert Zn and Mn c o n c e n t r a t i o n s measured on the AA i n the f i r s t year (1985) to 64 e q u i v a l e n t values f o r the ICP (Appendix D). The equations were used i n the r e t r a n s l o c a t i o n study to compare f i r s t year values measured on the AA to f o l i a r values from the second year (1986) measured on the ICP. A comparison was made between AA and ICP fo r the N a t i o n a l Bureau of Standard samples i n the percentage recovery of Ca, Mg, K, Mn, Zn, and A l from f o l i a g e . T h i s i n f o r m a t i o n i s presented i n Appendix E. Boron was determined by dry ashing, followed by c o l o r i m e t r i c a n a l y s i s by the azomethine H method of Gaines and M i t c h e l l (1979). T o t a l sulphur was determined using a F i s h e r Model 475 Sulphur Analyzer using the procedure d e s c r i b e d by Guthrie and Lowe (1984). Sulphate-sulphur was e x t r a c t e d from the f o l i a g e a c c ording to the procedure o u t l i n e d i n Appendix B3 and determined according to the method d e s c r i b e d by Kowalenko and Lowe (1972) (Appendix B3). The i d e a that o n l y a p o r t i o n of the t o t a l l e v e l of an element may be p h y s i o l o g i c a l l y a c t i v e l e d to the need to determine a c t i v e i r o n , and w a t e r - e x t r a c t a b l e z i n c and manganese. The concept of a m e t a b o l i c a l l y a c t i v e f r a c t i o n of i r o n was i n v e s t i g a t e d by Oserkowsky (1933). His procedure f o r the det e r m i n a t i o n of a c t i v e i r o n , s l i g h t l y modified by B a l l a r d (1981), i s d e s c r i b e d i n Appendix B4. The p h y s i o l o g i c a l l y a v a i l a b l e z i n c was determined using a modified procedure of 65 Cakmak and Marschner (1987) d e s c r i b e d i n Appendix B5. E x t r a c t a b l e manganese was determined using a modified water e x t r a c t i o n method of Memon and Yatazawa (1982) (Appendix B6). The c e l l u l a r f r a c t i o n s i n f o l i a g e were separated using the method of Memon and Yatazawa (1984) s l i g h t l y modified by t h i s author d e s c r i b e d i n Appendix F. T o t a l z i n c and manganese were determined i n the d i f f e r e n t f r a c t i o n s using the Parkinson and A l l e n d i g e s t i o n . Three r e p l i c a t e s were done f o r each sample. One experiment compared z i n c and manganese l e v e l s i n d i f f e r e n t f r a c t i o n s from the high f o l i a r z i n c treatment and the high s o i l manganese treatment. These samples were from c u r r e n t year's f o l i a g e i n the f i r s t season f o l l o w i n g f e r t i l i z a t i o n (1986) from s i t e 5. The second experiment compared z i n c and manganese l e v e l s i n the f o l i a g e from d i f f e r e n t s p e c i e s using the c u r r e n t year's f o l i a g e of 1987 from s i t e 5. F. Scanning E l e c t r o n Mlcroprobe In a d d i t i o n to comparing the n u t r i t i o n of s p e c i e s using t o t a l n u t r i e n t l e v e l s , e x t r a c t a b l e l e v e l s and c e l l u l a r f r a c t i o n s , an attempt was made to measure the n u t r i e n t s i n the f o l i a g e of d i f f e r e n t s p e c i e s in situ. T h i s was done using e l e c t r o n probe microanalyzer (EPMA) with scanning e l e c t r o n microscopy (SEM). The t i s s u e was f i x e d and prepared p a r t l y i n the f i e l d and the l a b o r a t o r y a c c o r d i n g to the method d e s c r i b e d i n Appendix G. The SEM-EPMA i n the Department of M e t a l l u r g y at the U n i v e r s i t y of 66 B r i t i s h Columbia was used. However, the r e s o l u t i o n of EPMA i s not f i n e enough to measure m i c r o n u t r i e n t s i n the range of ug s ~ % . A l t e r n a t i v e l y , an attempt was made to have the a n a l y s i s done on a scanning proton mlcroprobe (SPM); however, the p r i c e of the a n a l y s i s was c o s t - p r o h i b i t i v e . G. S o i l Sample P r e p a r a t i o n and A n a l y s i s The mineral s o i l and f o r e s t f l o o r samples were a i r - d r i e d at room temperature (22*C). The s o i l samples were s i e v e d through a 2.0-mm s t a i n l e s s s t e e l mesh s i e v e . F o r e s t f l o o r samples were ground i n a Waring blender to pass a 20-mesh s i e v e . The samples were analyzed f o r pH, c a t i o n exchange c a p a c i t y , t o t a l N (TH), e x t r a c t a b l e NH*~ (EXTN), P, K, Ca, Mg, Mn, Zn, Cu, and Fe. S o i l pH was measured i n water and 0.01 M CaCls, using a g l a s s e l e c t r o d e pH meter. The s o i l to s o l u t i o n d i l u t i o n r a t i o s were 1:2 f o r mineral s o i l s and 1:8 f o r org a n i c s o i l s . C a t i o n exchange c a p a c i t y was determined using the sodium c h l o r i d e method. T h i s i s an unbuffered s o l u t i o n e n a b l i n g e v a l u a t i o n of the c a t i o n exchange c a p a c i t y of the s o i l at i t s i n h e r e n t pH. T o t a l N was determined using semi-micro K J e l d a h l d i g e s t i o n f o l lowed by c o l o r i m e t r i c d e t e r m i n a t i o n using the Autoanalyzer. A v a i l a b l e MHA* was assayed using an e x t r a c t i o n i n 2% KaSO* followed by c o l o r i m e t r i c d e t e r m i n a t i o n using the Autoanalyzer. A v a i l a b l e P, K, Ca, Mg, Mn, Zn, Cu, and Fe were 67 e x t r a c t e d using the Mehlich 3 s o i l t e s t e x t r a c t a n t (Mehlich 1984) as modified by B a l l a r d (Appendix H). The e x t r a c t a n t s were analyzed by ICP. H. Measurement of F e r t i l i z e r Response Response to f e r t i l i z a t i o n was measured using v a r i o u s growth parameters: height increment r a t i o , shoot l e n g t h increment r a t i o , f o l i a r mass per shoot and f o l i a r n u t r i e n t c o n c e n t r a t i o n . Where there was a s i g n i f i c a n t d i f f e r e n c e i n f o l i a r mass: f o l i a r n u t r i e n t c o n c e n t r a t i o n , n u t r i e n t content per shoot and f o l i a r mass per shoot were evaluated together as a measure of growth r e s p o n s e I I N I ' I I K t h e v« i : l :or ; m e t h o d off T i m n i K r a n d S t o n e (1.978) (Figure 6 ) . R e s u l t s of the "complete-Zn-Mn" treatment were presented using v e c t o r a n a l y s i s on a r e l a t i v e b a s i s . T h i s permits comparison of v a r i o u s elements together on one graph. The ascending order of elements along a r e l a t i v e u n i t weight l i n e i n d i c a t e s the degree of d e f i c i e n c y f o r these elements (Timmer and Morrow 1984). Otherwise n u t r i e n t c o n c e n t r a t i o n was used alone. I n t e r p r e t a t i o n of f o l i a r n u t r i e n t c o n c e n t r a t i o n s was done using the summary of B a l l a r d and C a r t e r (1986) presented i n Appendix I. Growth and n u t r i e n t responses were evaluated i n terras of the s t a t i s t i c a l s i g n i f i c a n c e of the treatments from t h e i r r e s p e c t i v e c o n t r o l s . 68 R e l a t i v e shoot increment and height increment were a l s o used to evaluate growth response using the pretreatment increment method of B a l l a r d and Majid (1985). The use of pretreatment increment allows adjustment f o r s i t e as w e l l as stand s t r u c t u r e d i f f e r e n c e s . Shoot growth as well as height growth response were expressed as the r a t i o of p o s t - f e r t i l i z a t i o n to p r e - f e r t i l i z a t i o n shoot and height increment. Af/Bf compared with Ac/Be where: A • increment a f t e r f e r t i l i z a t i o n B = increment before f e r t i l i z a t i o n f - f e r t i l i z e d c = c o n t r o l The r a t i o Af/Bf i s an index of f e r t i l i z e r response as well as environmental e f f e c t s , whereas Ac/Be i s an index of only environmental i n f l u e n c e . T h e i r d i f f e r e n c e p r o v i d e s a measurement of s o l e l y the treatment e f f e c t ( B a l l a r d and Majid 1985). Shoot l e n g t h increment may sometimes be an i n d i c a t o r of f u t u r e volume growth. Barker (1978) found a c o r r e l a t i o n (r=0.95) of the d i f f e r e n c e i n shoot increment (between the f e r t i l i z e d and c o n t r o l i n the previous year) with the d i f f e r e n c e i n volume increment (between the f e r t i l i z e d and the c o n t r o l ) i n the c u r r e n t year. 69 I. S t a t i s t i c a l A n a l y s i s A l l s t a t i s t i c a l t e s t s f o r s i g n i f i c a n c e were performed using both parametric and non-parametric methods. There were three groups of data s u b j e c t e d to s t a t i s t i c a l t e s t s . Because each f e r t i l i z e r treatment had i t s own c o n t r o l , the n u l l hypothesis of no treatment e f f e c t could be evaluated by parametric two-sample t - t e s t s . If the d i f f e r e n c e s between the v a r i a n c e s were more than t h r e e - f o l d , the r e s u l t s were checked using the non-parametric Mann-Whitney t e s t . For the experiment i n v o l v i n g comparison of f o l i a r n u t r i e n t c o n c e n t r a t i o n s between s p e c i e s , the n u l l hypothesis which was t e s t e d was that there were no d i f f e r e n c e s i n f o l i a r n u t r i e n t c o n c e n t r a t i o n s between s p e c i e s . The n u l l hypotheses was t e s t e d by a one-way a n a l y s i s of v a r i a n c e . D i f f e r e n c e s between means were analyzed using the parametric Tukey m u l t i p l e comparison t e s t . The Tukey t e s t allows f o r the unbalanced c o n d i t i o n and i s g e n e r a l l y p r e f e r r e d to the other m u l t i p l e range t e s t s (Wilkinson 1988). I f the v a r i a n c e s were not homogeneous, as determined by B a r t l e t t ' s t e s t , the s i g n i f i c a n c e of d i f f e r e n c e between means was checked using the K r u s k a l - W a l l i s t e s t as a non-parametric a n a l y s i s of v a r i a n c e , and the Mann-Whitney t e s t as a m u l t i p l e comparison t e s t . The p r o b a b i l i t y t a b l e used f o r the Mann-Whitney t e s t was adjusted using the B o n f e r r o n i procedure (Wilkinson 1988) to take i n t o account the comparison of more than one p a i r of 70 means. Without such an adjustment, there would be i n c r e a s e d p r o b a b i l i t y of making a type I e r r o r when using a two-sample t e s t f o r more than two means f o r reasons d e s c r i b e d by Zar (1984). The data examining r e t r a n s l o c a t i o n i n the f o l i a g e were analyzed using the parametric p a i r e d t - t e s t and the non- parametric e q u i v a l e n t t e s t , the Wilcoxon signed rank t e s t . R e l a t i o n s h i p s between n u t r i e n t s , between n u t r i e n t s and growth, and between growth parameters were i n v e s t i g a t e d using c u r v i l i n e a r r e g r e s s i o n methods. A l l t - t e s t s , ANOVAs and r e g r e s s i o n s were evaluated at the 5X l e v e l of s i g n i f i c a n c e . 71 CHAPTER 4. RESULTS A. COMPARATIVE NUTRITION A comparison of n u t r i t i o n was made among s p e c i e s o c c u r r i n g i n the same stands (Table 8) to determine whether the n u t r i t i o n a l c h a r a c t e r i s t i c s of hemlock are s p e c i e s - or s i t e - r e l a t e d . 1. T o t a l L e v e l s Hemlock tends to have lower f o l i a r Zn c o n c e n t r a t i o n s compared with D o u g l a s - f i r , a m a b i l i s f i r and white pin e . But i t has higher f o l i a r manganese c o n c e n t r a t i o n s compared to a l l the other s p e c i e s examined. 2. E x t r a c t a b l e Zn and Mn Although not examined i n t h i s study, i t i s i n f e r r e d that s u b s t a n t i a l l y d i f f e r e n t c o n c e n t r a t i o n s of e x t r a c t a b l e n u t r i e n t s i n d i f f e r e n t s p e c i e s on the same s i t e may be i n d i c a t i v e of d i f f e r e n t p h y s i o l o g i c a l requirements. The water-soluble f r a c t i o n of Zn has been shown to repr e s e n t the p h y s i o l o g i c a l l y a c t i v e component by Rahimi and Shropp (1984) and Cakmak and Marschner (1987). Water-soluble Mn has been shown by Memon et al. (1980) to exclude the f r a c t i o n of Mn bound i n the c e l l wall and the l a t t e r may be p h y s i o l o g i c a l l y i n a c t i v e . 72 T a b l e 8 . T o t a l f o l i a r Zn and Mn, w a t e r - s o l u b l e Mn ( A M N ) , and a c t i v e Zn (AZN) f o r d i f f e r e n t s p e c i e s i n t h e same s t a n d s . W i t h i n a c o l u m n means w i t h d i f f e r e n t l e t t e r s a r e s i g n i f i c a n t l y d i f f e r e n t a t t h e 5% l e v e l u s i n g A N O V A . V a l u e s a r e n u t r i e n t m a s s / d r y m a t t e r i n ug g - 1 - . S H E 1 1986 MEAN SPECIES D o u g l a s - f i r A m a b i l i s f i r Hemlock ZN 22 .3a 17.2a 9 . 7b MN 473a 571a 1139b AZN 19 ,0a 11.5b 8.8b AMN 755a 842ab 1218b S I T E 4-1987 MEAN SPECIES ZN MN D o u g l a s - f i r 17.2a 515a A m a b i l i s f i r 16.0 675a Hemlock 11.7b 1545b S I T E 2-1986 MEAN SPECIES Red c e d a r White p i n e Hemlock S I T E 3-1986 MEAN ZN 12.6a 29 .Ob 11.1a MN 246a 375a 1932b AZN 4.3a 20.7b 12 . l c AMN 117a 439a 1948b S i t e 5-1987 MEAN SPECIES ZN MN D o u g l a s - f i r 14.9 265a A m a b i l i s f i r 17.2a 415b Y e l l o w c e d a r 11.9b 23a Hemlock 11.8b 1243c SPECIES ZN MN A m a b i l i s f i r Hemlock 18.3a 11.0b 916a 1505b S I T E 4-1986 MEAN SPECIES ZN MN D o u g l a s - f i r 20.0a 713a A m a b i l i s f i r 19.1a 735a Hemlock 8.7b 1428b S I T E 5-1986 MEAN SPECIES D o u g l a s - f i r A m a b i l i s f i r Hemlock ZN 18 . 0a 10.3b 11.4b MN 472a 220b 1308c 73 It was hypothesized that although d i f f e r e n t s p e c i e s may have d i f f e r e n t t o t a l f o l i a r l e v e l s of a n u t r i e n t , the p h y s i o l o g i c a l l y a c t i v e f r a c t i o n may be s i m i l a r . T h e r e f o r e , to examine t h i s hypothesis the a c t i v e Zn and water-so l u b l e l e v e l s of Mn, and t o t a l f o l i a r l e v e l s of Zn and Mn were compared among s p e c i e s on s i t e s 1 and 2 (Table 8 ) . On s i t e 1, D o u g l a s - f i r and am a b i l i s f i r have higher t o t a l Zn than hemlock. D o u g l a s - f i r has higher a c t i v e Zn than hemlock, but a m a b i l i s f i r has s i m i l a r l e v e l s of a c t i v e Zn to hemlock. On s i t e 2 , white pine had higher t o t a l and a c t i v e Zn than hemlock, which i n t u r n had higher l e v e l s than red cedar. On s i t e 1, hemlock had higher t o t a l and water-soluble Mn l e v e l s as compared to D o u g l a s - f i r and a m a b i l i s f i r . On s i t e 2 hemlock had higher t o t a l and water-soluble Mn c o n c e n t r a t i o n s than white pine and red cedar. 3 . C e l l u l a r F r a c t i o n s of F o l i a g e L e v e l s of Zn and Mn were compared between c o n t r o l and t r e a t e d samples, and between s p e c i e s . The data appear i n Appendix J . I t was hypothesized t h a t although d i f f e r e n t s p e c i e s may have d i f f e r e n t t o t a l f o l i a r l e v e l s of Zn and Mn the l e v e l s i n the d i f f e r e n t c e l l u l a r f r a c t i o n s which rep r e s e n t d i f f e r e n t p h y s i o l o g i c a l processes may be s i m i l a r . The f r a c t i o n s i d e n t i f i e d by Memon (1984) were A, corresponding to c e l l w a l l and d e b r i s ; B, c h l o r o p l a s t s ; C, mitochondria; D, ribosomes; and E, vacuoles. In the present study, f r a c t i o n s A and B corresponded to Memon's A and B, C contained c h l o r o p l a s t s , D corresponded to the 74 m i t o c h o n d r i a l f r a c t i o n , and E contained the ribosomal and v a c u o l a r f r a c t i o n s . For Zn, the c e l l f r a c t i o n s from the high Zn treatment had higher l e v e l s of Zn than the c o n t r o l f r a c t i o n s ( F igure 10). The s i t u a t i o n was s i m i l a r f o r Mn i n that f o r a l l f r a c t i o n s the Mn treatment had the higher l e v e l of Mn compared to the c o n t r o l ( F i g u r e 11). In comparing Zn and Mn treatments accumulation o c c u r r e d i n two d i f f e r e n t f r a c t i o n s . Zn tended to accumulate i n f r a c t i o n D and Mn tended to accumulate i n f r a c t i o n E. In a comparison of u n f e r t i l i z e d t r e e s , there was a d i f f e r e n c e among s p e c i e s f o r Mn c o n c e n t r a t i o n s i n c e l l u l a r f r a c t i o n s ( F i g u r e 12). Hemlock had higher Mn l e v e l s i n f r a c t i o n s A, B, C, D and E. Yellow cedar had the lowest Mn l e v e l s of any s p e c i e s i n f r a c t i o n s A, B, C, D and E. There was a predominance f o r Zn to accumulate i n f r a c t i o n D f o r a l l s p e c i e s (Figure 13). D o u g l a s - f i r had the h i g h e s t Zn c o n c e n t r a t i o n i n the D f r a c t i o n compared to the other s p e c i e s . 75 F i g u r e 1 0 . Z i n c c o n c e n t r a t i o n s i n d i f f e r e n t c e l l u l a r f r a c t i o n s f r o m t h e c u r r e n t y e a r ' s f o l i a g e ( 1 9 8 6 ) o f t h e c o n t r o l t r e a t m e n t a n d h i g h f o l i a r Z n t r e a t m e n t f r o m s i t e 5 i n t h e f i r s t y e a r o f t r e a t m e n t . F r a c t i o n A = c e l l w a l l and d e b r i s , B + C = c h l o r o p l a s t s , D = m i t o c h o n d r i a , E = r i b o s o m e s and v a c u o l a r c o n t e n t s . 76 3000 Manganese us s-- Sample 1 I C o n t r o l 600 k g Mn ha" 1 - F i g u r e 1 1 . M a n g a n e s e c o n c e n t r a t i o n s i n d i f f e r e n t c e l l u l a r f r a c t i o n s f r o m t h e c u r r e n t y e a r ' s f o l i a g e ( 1 9 8 6 ) o f t h e c o n t r o l t r e a t m e n t a n d h i g h s o i l Mn t r e a t m e n t f r o m s i t e 5 i n t h e f i r s t y e a r o f t r e a t m e n t . F r a c t i o n A = c e l l w a l l and d e b r i s , B + C = c h l o r o p l a s t s , D = m i t o c h o n d r i a , E = r i b o s o m e s and v a c u o l a r c o n t e n t s . 77 Manganese,ug g _ i 1400 | : 1200 - 1000 - 800 - 600 - I" 400 - A B O D E Fractions Species I 1 Hemlock • ! Douglas-fir CZ! Amabil is fir ZH Yellow cedar F i g u r e 1 2 . M a n g a n e s e c o n c e n t r a t i o n s i n d i f f e r e n t c e l l u l a r f r a c t i o n s f r o m t h e c u r r e n t y e a r ' s f o l i a g e i n 1 9 8 7 o f u n f e r t i l i z e d t r e e s o f d i f f e r e n t s p e c i e s on s i t e 5 . F r a c t i o n A = c e l l w a l l and d e b r i s , B + C = c h l o r o p l a s t s , D = m i t o c h o n d r i a , E = r i b o s o m e s and v a c u o l a r c o n t e n t s . 78 120 Zinc Ms s - i 100 - I J Hemlock 8peciea I Douglas-f i r Mill Amabil is f ir Yellow cedar F i g u r e 13. Z i n c c o n c e n t r a t i o n s i n d i f f e r e n t c e l l u l a r f r a c t i o n s from the c u r r e n t year's f o l i a g e i n 1987 of u n f e r t i l i z e d t r e e s of d i f f e r e n t s p e c i e s on s i t e 5. F r a c t i o n A = c e l l w a l l and d e b r i s , B + C = c h l o r o p l a s t s , D = m i t o c h o n d r i a , E = ribosomes and v a c u o l a r c o n t e n t s . 79 In f r a c t i o n s A, B, and C hemlock tended to have lower l e v e l s than the other s p e c i e s . In f r a c t i o n E, hemlock had lower Zn than D o u g l a s - f i r and a m a b i l i s f i r . In a l l f r a c t i o n s D o u g l a s - f i r tended to have higher Zn c o n c e n t r a t i o n s than hemlock. B. F e r t i l i z a t i o n Experiments 1. N u t r i e n t and Growth Responses C o n v e n t i o n a l l y vector a n a l y s i s has been a p p l i e d to s p e c i e s with determinate shoot growth and has been expressed as mass per f i x e d number of needles. For hemlock, the q u e s t i o n has a r i s e n whether f o l i a r mass should be expressed on the b a s i s of a f i x e d number of needles or on a per shoot b a s i s . Hemlock has both determinate and indeterminate shoot growth, hence i t s s p e c i e s name heterophylla, hetero meaning d i f f e r e n t , and phylla meaning lea v e s (Harlow and Harrar 1958). The d i s t a l needles on a 1-year- o l d shoot are s h o r t e r than the proximal needles (Owen and Holder 1973). The proximal needles are the r e s u l t of determinate shoot growth. These needles were formed i n the bud the previous year, and t h e r e f o r e t h e i r number would depend upon the environmental c o n d i t i o n s of the previous year, but t h e i r mass would r e f l e c t the c u r r e n t year's environment. The needles formed at the d i s t a l p a r t of the shoot are the r e s u l t of indeterminate shoot growth which means that they were i n i t i a t e d and elongated i n the same 80 year. T h e r e f o r e , both t h e i r number and mass would r e f l e c t the c u r r e n t year's environmental c o n d i t i o n s . It appears that f o l i a r mass per shoot would be an ap p r o p r i a t e parameter i n which to express growth f o r two reasons. F i r s t l y , f o l i a r mass per shoot i s a f u n c t i o n of both number of needles per shoot and mass per needle. T h e r e f o r e , changes i n e i t h e r of the two components would be r e f l e c t e d i n f o l i a r mass per shoot. Secondly, from r e g r e s s i o n a n a l y s i s there were s i g n i f i c a n t r e l a t i o n s h i p s between c u r r e n t height increment and f o l i a r mass per shoot (Figure 14 and Table 9 ) . The r e l a t i o n s h i p between f o l i a r mass per shoot and c u r r e n t height increment was p o s i t i v e and l i n e a r , but i n some cases the r e l a t i o n s h i p was c u r v i l i n e a r . The c u r v i l i n e a r r e l a t i o n s h i p i n d i c a t e s t hat a maximum p o i n t was reached beyond which an i n c r e a s e i n f o l i a r mass per shoot was a s s o c i a t e d with a r e d u c t i o n i n height increment. In cases where the change i n f o l i a r mass per shoot and f o l i a r c o n c e n t r a t i o n were determined to be s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l s , the response was assessed using vect o r a n a l y s i s . Otherwise, n u t r i e n t c o n c e n t r a t i o n was used alone. The n u t r i e n t and growth data f o r the f e r t i l i z a t i o n t r i a l s f o r a l l s i t e s and years are presented i n Appendix L. 81 F i g u r e 1 4 . S c a t t e r p l o t s o f t h e f o l i a r mass p e r s h o o t f o r s i t e 1 s e c o n d y e a r s ( 1 9 8 6 ) ( B ) . h e i g h t i n c r e m e n t v e r s u s t h e i n t h e f i r s t ( 1 9 8 5 ) ( A ) and 82 Table 9. Equations and c o r r e l a t i o n c o e f f i c i e n t s (R a) f o r the height increment (y) - f o l i a r mass per shoot (x) r e l a t i o n s h i p s f o r each s i t e . Refer to Appendix K f o r a d d i t i o n a l s c a t t e r p l o t s . 1 1986 y = 171.Ox - 117.Ox* 0.59 2 1986 y « 291.Ox - 440.0x a 0.38 K . l 3 1986 y = 154.Ox - 151.0x a 0.32 K.2 4 1986 y » 160.Ox - 71.0x a 0.55 K.3 4 1987 y • 16.0 + 163.Ox - 82.0x a 0.49 K.4 5 1986 y = 204.Ox - 143.0x a 0.43 K.5 5 1987 y » 206.Ox - 124.0x a 0.49 K.6 83 a. Zi n c i . F o l i a r Z i n c Treatments In the f i r s t growing season f o l l o w i n g f o l i a r z i n c a p p l i c a t i o n s , an i n c r e a s e i n uptake of f o l i a r z i n c o c c u r r e d . T h i s was true f o r a l l s i t e s and a l l l e v e l s of a p p l i c a t i o n (Table 10). In the second growing season f o l l o w i n g f o l i a r z i n c a p p l i c a t i o n s , f o l i a r z i n c l e v e l s were s t i l l e l e v a t e d on s i t e s 1 and 2 from treatment 5 (3600 mg Zn L~M, on s i t e 3 from treatment 4 (360 mg Zn L - 1 ) , and on s i t e 5 from treatment 15 (2700 mg Zn L-M (Table 10). On s i t e 2 i n the f i r s t year there was an i n c r e a s e i n f o l i a r mass and f o l i a r Zn from treatment 4 (360 mg Zn L -*-), which i m p l i e s a growth response (Table 10 and Fig u r e 15A) ( s h i f t i n C d i r e c t i o n ) . Conversely a r e d u c t i o n i n mass and i n c r e a s e i n f o l i a r Zn from treatment 5 (3600 mg Zn L~ x) i n d i c a t e a Zn t o x i c i t y ( F i g u r e 15A) ( s h i f t i n E d i r e c t i o n ) . On s i t e 3 there was an i n c r e a s e i n f o l i a r mass and f o l i a r Zn a f t e r the second year, from treatment 4 (360 mg Zn L - 1 ) , i n d i c a t i n g a growth response ( F i g u r e 15B) ( s h i f t i n C d i r e c t i o n ) . On s i t e 5 i n the f i r s t year, there was a decrease i n f o l i a r mass and i n c r e a s e i n f o l i a r Zn from treatment 13 (360 mg Zn L - i ) , suggesting a Zn t o x i c i t y ( F igure 15C) ( s h i f t i n E d i r e c t i o n ) . 84 T a b l e 10. F o l i a r z i n c ( u g g - 1 ) c o n c e n t r a t i o n r e s p o n s e and f o l i a r mass p e r s h o o t ( g ) g r o w t h r e s p o n s e t o f o l i a r a p p l i c a t i o n s o f z i n c i n t h e f i r s t and s e c o n d y e a r s f o l l o w i n g t r e a t m e n t s . V a l u e s i n p a r e n t h e s e a r e t h e s t a n d a r d d e v i a t i o n . An (*) i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e a n d n . s . d . i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e a t t h e 5% l e v e l b e t w e e n a t r e a t m e n t and i t s r e s p e c t i v e s u l f u r c o n t r o l . S i t s 1 S i t e 3 - • F i r s t Tear Second Tear Second Tear T r e a t a e n t Za Kaas Za Haaa T r e a t a e a t Zn Haaa — u s *"*•- — t — —ft I"*- — s — — u s »-*•• — 5 — 360 i ; Zn [,->• 38 .1 0.18 13.4 0.264 360 as Za l"- 21.3 0.233 (46 .3) (0.154) (2.2) (0 .193) (2 .4 ) (0.125) 177 >; S (.-"• 177 as S L-» 10.5 ' 0.202 11.3 0.202 14.4 0.171 (3 .* ) (0 .064) (3.3) (0 .117) C3.7) (0.039) * a . a . d . a . a . d . a . a . d . * • 3600 » c Za !.-«• 3600 as Zn f » 19.1 0.193 1*9.7 0.241 15.2 0.206 (3 .3) (0.081) (46 .S) 1770 a t S L- 1 (0.154) (5.2) (0 .149) 1770 as S L-* 17.4 0.172 7.9 0 .208 11.1 0.140 ( 3 . 4 ) (0.103) / ' ( 2 .1 ) (0.076) (3.5) (0 .036) a . a . d . a . a . d . a . s . d . * a . a . d . Tab l e 10 ( c o n t i a u e d ) . S i t e 2 F i r s t Tear Second Tear T r e a t a e n t Za Haaa Zn Haaa — v i *"*— — t — —us «""•- ' ~ t - 360 as Za t " » 38 . 3 0.153 11.5 0.133 (21 .3) (0 .034) (5.6) (0.063) 177 a t S L-* 6.7 0.107 8.8 0.123 (2 .3 ) (0 .019) ( 3 . 4 ) (0 .039) • * o . a . d . a . a . d . 3600 a t Zn L~* 73.0 0.077 19.6 0.131 (99 .0) (0 .049) (3.8) (0 .092) 1770 a t S L-» 3.3 0.118 11 .4 0.163 ( 2 . 8) (0 .042) (2.7) (0 .113) • • * a . a . d . Table 10 (concluded) Site 4 F i r s t Year Treatment Zn Kass —VI 8"1 --% — 360 mg Zn L _ l 18.3 0.263 (6.9) (0.156) 177 mg S L" 1 8.2 0.216 (3.3) (0.231) : * n.s.d. 1800 mg Zn L" 1 26.2 0.299 (10.4) (0.082) 883 mg S L" 1 9.4 0.267 (3.1) (0.071) * n.s.d. 2700 mg Zn L~ l 48.6 0.171 (25.1) (0.136) 1325 mg S L" 1 6.4 0.136 (2.1) (0.063) * n.s.d. Second Year Zn "Pg g _ l- 10.2 (2.6) 8.5 (3.0) n.s.d. 8.3 (2.6) 8.5 (3.1) n.s.d. 9.4 (3.0) 9.7 (3.5) n.s.d. Mass — g — 0.421 (0.340) 0.327 (0.174) n.s.d. 0.371 (0 .266) 0.459 (0.277) n.s.d. 0 .368 (0.293) 0.515 (0.335) n.s.d. Site 5 F i r s t Year Second Year Treatment Zn Haas Zn Hasa — Mg g" 1 " g — —vs g~ 1-- ~ g ~ 360 mg Zn L - t 79.3 0.184 11.9 0.306 (30.1) (0.078) (2.9) (0.152) 177 mg S L~ l 11.2 0.291 10.5 0.278 (1.9) (0.052) (4.6) (0.110) • * n . s". d . n.s.d 1800 mg Zn L - 1 100.9 0.173 15 .4 0 .292 (31.8) (0.078) (4.9) (0.104) 883 mg S L - 1 10.1 0.198 12.5 0.301 (3.3) (0.071) (5.1) (0.151) * n.s.d. n.s.d. n.s.d. 2700 mg Zn L~ l 132.3 0.171 16.5 0 .314 (30.5) (0 .093) (4.5) (0.170) 1325 mg S L - 1 10.5 0.174 12.4 0 .308 (1.9) (0.052) (3.5) (0.165) 86 Foliar Mass 100 0.1 0.2 4 6 Zinc Content 0.1S Foliar Mass Z I n c C o n c e n t r a t I o n 140 120 100 - J.25 Foliar Mass 0.15 0.2 10 15 Zinc Content F i g u r e 1 5 . V e c t o r d i a g r a m s o f g r o w t h r e s p o n s e t o f o l i a r a p p l i e d z i n c . ( A ) F i r s t y e a r i n 1985 o n s i t e 2 , ( B ) s e c o n d y e a r i n 1986 on s i t e 3 , and ( C ) f i r s t y e a r i n 1986 on s i t e 5 . The x - a x i s i s pg Z n p e r s h o o t , t h e y - a x i s i s ug Zn g - t and t h e d a s h e d l i n e i s f o l i a r mass p e r s h o o t i n g r a m s . The a r r o w i n d i c a t e s t h e d i r e c t i o n o f t h e r e s p o n s e . T r e a t m e n t s a r e i n mg Zn L - 1 - . 87 There was no t o x i c i t y found with g r e a t e r a p p l i c a t i o n s of f o l i a r Zn. There were no s i g n i f i c a n t e f f e c t s of Zn a p p l i c a t i o n on f o l i a r mass per shoot i n the f i r s t year on s i t e 1 from treatment 4 (360 mg Zn L~M, on s i t e 4 from treatments 13 (360 mg Zn L~M, 14 (1800 mg Zn L - M , and IS (2700 mg Zn L -*-), and s i t e 5 from treatments 14 (1800 mg Zn L~M and 15 (2700 mg Zn L~"M but i n c r e a s e d uptake of Zn o c c u r r e d , i n d i c a t i n g l u xury consumption on those s i t e s . i i . S o i l Z i n c Treatments A d i f f e r e n t p a t t e r n of response was found f o r the z i n c s o i l treatments. No response occurred i n the f i r s t growing season f o l l o w i n g f e r t i l i z a t i o n on a l l s i t e s . However, i n the second growing season, n u t r i e n t c o n c e n t r a t i o n response occurred from treatments 1 (10 kg Zn ha~ x) and 2 (50 kg Zn ha~ x) on s i t e 1, treatment 2 (50 kg Zn ha""M on s i t e 3, and from treatment 3 (200 kg Zn h a ~ l ) on s i t e 5 (Table 11). Since there were no i n c i d e n t s of s i g n i f i c a n t i n c r e a s e s i n f o l i a r mass per shoot, these r e s u l t s i n d i c a t e l u x u r y consumption of Zn on these s i t e s . On s i t e s 4 a p o s i t i v e growth response occurred i n the second year from treatment 3 (200 kg Zn ha~*x) (Figure 16) ( s h i f t i n C d i r e c t i o n ) . 88 T a b l e 11. F o l i a r z i n c ( p g g - 1 ) c o n c e n t r a t i o n r e s p o n s e a n d f o l i a r mass p e r s h o o t ( g ) g r o w t h r e s p o n s e t o s o i l a p p l i c a t i o n s o f z i n c i n t h e s e c o n d y e a r f o l l o w i n g t r e a t m e n t . V a l u e s i n p a r e n t h e s e a r e t h e s t a n d a r d d e v i a t i o n . A n (*) i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e a n d n . s . d . i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e . a t t h e 5% l e v e l b e t w e e n a t r e a t m e n t and i t s r e s p e c t i v e s u l f u r c o n t r o l . S i t e 1 T r e a t m e n t Zn Mass — s - 3 - - ~ s — 10 kg Zn h a " 1 13.3 0.248 (3.9) (0.206) 5 kg S h a - 1 9.9 0.213 : (2.4) (0.131) * n . s . d . 50 kg Zn h a " 1 17.2 0.221 (10.3) (0.120) 25 kg S h a " 1 11.4 0.343 (1.5) (0.231) * n . s . d . S i t e 3 50 kg Zn h a " 1 17.7 0.303 (10.0) (0.248) 25 kg S h a " 1 12.8 0.303 (4.3) (0.360) S i t e 4 T r e a t m e n t Zn Mass -ug g _ 1 - — S — 200 kg Zn h a " 1 11.2 0.364 (2.7) (0.195) 98 kg S h a " 1 6.4 0.199 (2.3) (0.084) S i t e 5 200 kg Zn h a " 1 15.2 0.258 (5.7) (0.212) 98 kg S h a - 1 8.8 0.272 (1.8) (0.108) * n . s . d . 89 F i g u r e 1 6 . V e c t o r d i a g r a m o f t h e s e c o n d y e a r g r o w t h r e s p o n s e i n 1 9 8 7 t o s o i l a p p l i e d z i n c on s i t e 4 f o r f o l i a r z i n c . The x - a x i s i s p g Zn p e r s h o o t , t h e y - a x i s i s pg Zn g - i and t h e d a s h e d l i n e i s f o l i a r mass p e r s h o o t i n g r a m s . The a r r o w i n d i c a t e s t h e d i r e c t i o n o f t h e r e s p o n s e . T r e a t m e n t s a r e i n kg Z n h a - 1 . 90 i i i . Comparison of F o l i a r Versus S o i l Treatments F o l i a r Zn treatments from a l l s i t e s were more e f f i c i e n t i n s u p p l y i n g the p l a n t with Zn ( F i g u r e 17). T h i s was c a l c u l a t e d as the change i n f o l i a r Zn c o n c e n t r a t i o n (of t r e a t e d minus c o n t r o l v alues) per gram of Zn a p p l i e d per t r e e . In a d d i t i o n , the i n c r e a s e i n f o l i a r Zn occurred i n the f i r s t year with the f o l i a r Zn a p p l i c a t i o n s whereas, i t was delayed u n t i l the second year with the s o i l Zn a p p l i c a t i o n s . The lower f o l i a r Zn treatment (360 mg Zn L - 1 ) was more e f f i c i e n t i n s u p p l y i n g the p l a n t with Zn than the higher f o l i a r Zn treatments on a l l s i t e s . b. Manganese i . F o l i a r Manganese Treatments The n u t r i e n t response to Mn tended to f o l l o w an opposite trend as compared with the Zn response. Response to f o l i a r - a p p l i e d Mn occurred i n the second year only on s i t e s 4 and 5, both to treatment 17 (4095 mg Mn L - 1 ) . On s i t e 4 the i n c r e a s e i n growth r e s u l t e d i n a manganese d i l u t i o n ( F i g u r e 18A) ( s h i f t i n A d i r e c t i o n ) , and on s i t e 5 the r e d u c t i o n i n growth and Mn c o n c e n t r a t i o n i s evidence of a manganese t o x i c i t y ( c o n c e n t r a t i o n e f f e c t , s h i f t i n F d i r e c t i o n ) (Figure 18B). 91 F i g u r e 1 7 . N u t r i e n t e f f i c i e n c y o f f o l i a r t r e a t m e n t s i n s u p p l y i n g t h e p l a n t w i t h Zn 2 7 0 0 and 3600 a r e i n mg 2 0 0 a r e i n kg Zn h a - 1 - . Zn c o n c e n t r a t i o n (pg g Zn v e r s u s s o i l Zn T r e a t m e n t s 3 6 0 , 1 8 0 0 , Z n L - 1 , and t r e a t m e n t s 1 0 , 5 0 , 100 and N u t r i e n t e f f i c i e n c y i s c h a n g e i n f o l i a r _ 1 ) p e r g ram o f Zn a p p l i e d p e r t r e e . 92 Foliar Maas M 1800 n g a n e s e C o n 1700 1600 1500 1400 0.4 A ^ 4 0 9 5 / — i I \ / <2>30 600 600 700 800 900 Manganese Content F i g u r e 1 8 . V e c t o r d i a g r a m s o f t h e s e c o n d y e a r g r o w t h r e s p o n s e i n 1 9 8 7 t o f o l i a r a p p l i e d Mn on s i t e 4 ( A ) a n d on s i t e 5 ( B ) f o r f o l i a r M n . The x - a x i s i s pg Mn p e r s h o o t , t h e y - a x i s i s pg Mn g _ l a n d t h e d a s h e d l i n e i s f o l i a r mass p e r s h o o t i n g r a m s . The a r r o w i n d i c a t e s t h e d i r e c t i o n o f t h e r e s p o n s e . T r e a t m e n t s a r e mg Mn L " 1 - . 93 i i . S o i l Manganese Treatments In c o n t r a s t , Mn n u t r i e n t c o n c e n t r a t i o n response to the s o i l Mn treatments occurred on a l l s i t e s at a l l l e v e l s of treatments except f o r treatment 4 (200 kg Mn h a - 1 ) on s i t e 4 (Table 12). T h i s response was s t i l l e vident i n the second growing season f o l l o w i n g manganese f e r t i l i z a t i o n f o r a l l manganese treatments and s i t e s . T h i s was l u x u r y consumption on s i t e s 1, and 2 from treatment 3 (200 kg Mn h a - M i n the f i r s t and second years, from treatment 3 (200 kg Mn h a " M on s i t e 3 i n the second year, on s i t e 4 from treatment 5 (400 kg Mn h a _ x ) i n the f i r s t and second years, from treatment 6 (600 kg Mn h a - 1 ) i n the f i r s t year, and on s i t e 5 from treatments 4 (200 kg Mn h a " 1 ) , 5 (400 kg Mn ha -*) and 6 (600 kg Mn h a - 1 ) i n the second year (Table 12) . Growth responses to s o i l - a p p l i e d manganese were obtained on s i t e s 4 and 5. On s i t e 4 a growth response was not detected u n t i l i n the second year from treatment 6 (600 kg Mn h a - 1 ) ( s h i f t i n C d i r e c t i o n ) (Figure 19A). With treatment 4 (200 kg Mn h a - M , a Mn t o x i c i t y was obtained ( s h i f t i n E d i r e c t i o n ) , but at the highest Mn l e v e l (treatment 6 (600 kg Mn h a ~ M ) , a p o s i t i v e growth response to manganese was obtained ( s h i f t i n C d i r e c t i o n ) . Response on s i t e 5 was d i f f e r e n t i n that a p o s i t i v e growth response to manganese was evident i n the f i r s t season. 94 T a b l e 1 2 . F o l i a r m a n g a n e s e (ug g - 1 ) c o n c e n t r a t i o n and f o l i a r mass p e r s h o o t ( g ) g r o w t h r e s p o n s e t o s o i l t r e a t m e n t s o f m a n g a n e s e . V a l u e s i n p a r e n t h e s e a r e t h e s t a n d a r d d e v i a t i o n . An (*) i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e and n . s . d . i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e a t t h e 5 % l e v e l b e t w e e n a t r e a t m e n t a n d i t s r e s p e c t i v e s u l f u r c o n t r o l . S i t e 1 F i r s t Year T r e a t m e n t Mn Hass -pg g _ l - — g — 200 kg Mn h a " 1 3008 0.360 (1298) (0.246) C o n t r o l 1002 0.283 (306) (0.127) * n . s . d . S i t e 2 200 kg Mn h a " 1 3142 0.145 (1470) (0.056) C o n t r o l 1449 0.143 (655) (0.049) * n . s . d . S i t e 3 T r e a t m e n t 200 kg Mn h a " 1 C o n t r o l Second Year Mn Mass -Pg g - 1 g ~ 2427 0.308 (441) (0.175) 1505 0.262 (287) (0.085) Second Year Mn Hass "Pg g " 1 " — g — 2971 0.348 (1278) (0.192) 1139 0.195 (395) (0.131) * n . s . d . 3875 0.181 (1429) (0.096) 1932 0.186 (589) (0.096) * n . s . d . Table 12 (concluded) Site 4 F i r s t Year Treatment Mn Mass "PS o~ l- — S — 200 kg Mn ha" 1 2026 0.270 (727) (0.096) 118 kg S ha~ l 1639 0.271 (597) (0.178) n.s.d. n.s.d. 400 kg Mn h a - 1 2802 0.352 (710) (0.388) 235 kg S ha" 1 1647 0.275 (604) (0.058) * n.s.d. 600 kg Mn h a - 1 3206 0.363 (1341) (0.284) 352 kg S ha" 1 1175 0.287 (214) (0.239) Second Year Mn Mass — pg g" 1 g — 2980 0.449 (918) (0.219) 1598 0.665 (412) (0.427) * * 3460 0.506 (780) (0.358) 1743 0.457 (471) (0.350) * n.s.d. 3737 0.646 (1237) (0.459) 1593 0.248 (365) (0.316) Site 5 F i r s t Year Treatment Hn Hass "PS g~ l" ~ g — 200 kg Mn ha" 1 1832 0.302 (510) (0.096) 118 kg S ha" 1 1083 0.193 (363) (0 :075) * * 400 kg Mn ha" 1 2172 0.250 (794) (0.134) 235 kg S ha" 1 1268 0.168 (408) (0.037) * * 600 kg Hn ha" 1 2933 0.367 (1021) (0.187) 352 kg S ha" 1 1240 0.250 (526) (0.210) Second Year Mn Mass --pg s - t - ~ g — 2487 0.317 (592) (0.089) 1359 0.291 (523) (0.156) * n.s.d. 2817 0.340 (651) (0.157) 1626 0.326 (632) (0.152) * n.s.d. 3518 0.316 (881) (0.103) 1301 0.314 (454) (0.180) * n.s.d. 96 (xWOO) Foliar Mass a t I o n 1' 1 — — 1 1 1 500 1000 1500 2000 2500 Manganese Content F i g u r e 1 9 . V e c t o r d i a g r a m s o f t h e g r o w t h r e s p o n s e t o s o i l a p p l i e d Mn f o r f o l i a r M n . ( A ) I n t h e s e c o n d y e a r ( 1 9 8 7 ) on s i t e 4 a n d ( B ) i n t h e f i r s t y e a r ( 1 9 8 6 ) on s i t e 5 . The x - a x i s i s pg Mn p e r s h o o t i n , t h e y - a x i s i s pg Mn g - 1 and t h e d a s h e d l i n e i s f o l i a r m a s s p e r s h o o t i n g r a m s . The a r r o w i n d i c a t e s t h e d i r e c t i o n o f t h e r e s p o n s e . T r e a t m e n t s a r e i n kg Mn h a " 1 . 97 A p o s i t i v e growth response to manganese occurred at a l l l e v e l s of treatment ( s h i f t i n C d i r e c t i o n ) (Figure 19B). c. Complete-Zn-Hn Treatment i . N u t r i e n t and Growth Responses A complete treatment was c a r r i e d out i n order to assess whether other n u t r i e n t d e f i c i e n c i e s were p r e s e n t . The r e s u l t s are summarized i n Table 13. On both s i t e s 4 and 5, growth and n u t r i e n t c o n c e n t r a t i o n responses were produced i n both the f i r s t and second years to the "complete -Zn -Mn" treatment. There was a p o s i t i v e growth response from the "complete-Zn-Mn" treatment on s i t e s 4 and 5 i n the f i r s t and second years (Table 13 and F i g u r e s 20 and 21) ( s h i f t i n C d i r e c t i o n ) . On s i t e 4 i n the f i r s t year (1986) the s e v e r i t y of n u t r i e n t d e f i c i e n c i e s ranked from most to l e a s t d e f i c i e n t were i n the order Zn, N, and B (Figure 20). On s i t e 5 i n the f i r s t year (1986) the s e v e r i t y of n u t r i e n t d e f i c i e n c i e s ranked from most to l e a s t d e f i c i e n t were i n the order B, Zn, AFe, Cu, Fe, and N (Figure 21A). In the second year the s e v e r i t y of n u t r i e n t d e f i c i e n c i e s were i n the order P, Zn and N ( F i g u r e 21B). Since there was a Zn response on s i t e s 4 and 5 to the "complete-Zn-Mn" treatment, the Zn response was a synergism to the a p p l i c a t i o n of other n u t r i e n t s ( s h i f t i n C d i r e c t i o n ) . 98 T a b l e 1 3 . F o l i a r n u t r i e n t c o n c e n t r a t i o n s and f o l i a r mass p e r s h o o t g r o w t h r e s p o n s e i n u n f e r t i l i z e d ( c o n t r o l ) t r e e s and t r e e s s u b j e c t e d t o t h e " c o m p l e t e - Z n - M n " t r e a t m e n t i n t h e f i r s t and s e c o n d y e a r s f o l l o w i n g t r e a t m e n t . An (*) i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e a t t h e 5% l e v e l o f s i g n i f i c a n c e . s Ca eg £ Site 4 1986 Complete -Zn-Mn 1.67 0.16 0.67 0.30 0.12 CO.26) (0.014) (0.108) (0.017) (0.022) Control 1.0 0.20 0.68 0.28 0.14 (0.06) (0.071) (0.195) (0.088) (0.062) Hn Fe Active Fe Zn HE S~ l Cu 1343 38.4 37.2 (400) (1.8) (2.4) 1428 25.0 32.0 (360) 17.2 3.5 33.2 (6.2) (0.6) (4.1) 8.7 3.2 24.5 (1.9) (1.8) S i t e 4 1987 Complete -Zn-Hn 1.03 0.18 0.56 0.21 0.10 (0.15) (0.024) (0.094) (0.053) (0.029) Control 0.94 0.12 0.57 0.23 0.09 (0.21) (0.041)(0.112) (0.088) (0.040) Site 5 1986 Complete -Zn-Mn Control 1.67 0.21 0.81 0.30 (0.22) (0.066) (0.115) (0.088) 1.11 0.18 0.78 0.32 (0.11) (0.052) (0.042) (0.061) 0 .136 (0 .052 0.14 (0.016 Site 5 1987 Complete -Zn -Mn 1.32 0.24 0.86 0.34 0.15 (0.15) (0.037) (0.225) (0.085) (0.040) Control 1.15 0.17 0.84 0.31 0.13 (0.017) (0.062) (0.177) (0.072) (0.024) 1259 51.2 (305) (10.6) 1545 48.8 (415) (15.4) 1064 46.4 34.8 (370) (9.1) (6.4) 1308 39.3 26.2 (531) (6.7) (5.3) 962 53.7 (387) (7.0) 1243 52.7 (666) (12.0) 11.1 2.5 (3.5) (0.6) 11.7 2.9 (4.6) (0.6) 15.5 4.0 45.8 (4.2) (0.6) (10.9) 11.4 3.1 28.4 (1.9) (0.6) (6.0) 14.7 3.6 (2.3) (0.7) 11.8 3.9 (3.6) (0.9) Table 13 ( c o n t i n u e d ) . N / P i H / P P / A l K/Ca Ca/Mg S i t e 4 1986 Complete -Zn -Mn C o n t r o l S i t e 4 1987 Complete -Zn -Mn C o n t r o l S i t e 5 1986 Complete -Zn -Mn C o n t r o l S i t e 5 1987 Complete -Zn -Mn C o n t r o l 10.4 10.3 4.3 2.2 2.6 5.1 6.2 4.5 2.5 2.0 5.9 6.4 7.6 4.6 7.3 3.1 7.8 10.3 6.1 6.9 5.5 8.2 6.9 7.1 4.9 3.3 4.4 2 . 7 2.2 2.3 2.7 2.4 2.6 2.7 2.2 2.1 2.2 2.3 2.3 2.4 S i t e 4 1986 Complete -Zn-Mn C o n t r o l S (cg g-1) 0.173 (0.029) 0.160 H/S 9.65 6.25 S i t e 5 1986 Complete -Zn-Mn C o n t r o l 0.114 (0.048) 0.124 (0.047) 14.65 8.95 1. C r i t i c a l value c a l c u l a t e d according to the formula. 100 Table 13 (concluded). F o l i a r Mass Per Shoot 1986 1987 g S i t e 4 Complete -Zn -Mn 0.804 0.635 (0.646) (0.346) C o n t r o l 0.200 0.280 (0.127) (0.145) S i t e 5 Complete -Zn -Mn 0.394 0.742 (0.162) (0.306) C o n t r o l 0.198 0.288 (0.054) (0.140) * * 101 Site 4 1986 F i g u r e 2 0 . V e c t o r d i a g r a m o f t h e f i r s t y e a r ( i n 1 9 8 6 ) g r o w t h r e s p o n s e t o t h e " c o m p l e t e - Z n - M n " t r e a t m e n t on s i t e 4 . The x - a x i s i s r e l a t i v e n u t r i e n t c o n t e n t p e r s h o o t , t h e y - a x i s i s r e l a t i v e n u t r i e n t c o n c e n t r a t i o n and t h e d a s h e d l i n e i s r e l a t i v e f o l i a r mass p e r s h o o t . The a r r o w i n d i c a t e s t h e d i r e c t i o n o f t h e ^ r e s p o n s e . The c o n t r o l = 1 0 0 . 102 F i g u r e 2 1 . V e c t o r d i a g r a m s o f t h e f i r s t ( i n 1 9 8 6 ) ( A ) and s e c o n d y e a r ( i n 1 9 8 7 ) ( B ) g r o w t h r e s p o n s e s t o t h e " c o m p l e t e - Z n - M n " t r e a t m e n t on s i t e 5 . The x - a x i s i s r e l a t i v e n u t r i e n t c o n t e n t p e r s h o o t , t h e y - a x i s i s r e l a t i v e n u t r i e n t c o n c e n t r a t i o n and t h e d a s h e d l i n e i s r e l a t i v e f o l i a r mass p e r s h o o t . The a r r o w i n d i c a t e s t h e d i r e c t i o n o f t h e r e s p o n s e . The c o n t r o l = 1 0 0 . 103 The Increase i n f o l i a r Zn suggests that a d d i t i o n a l Zn was r e q u i r e d with f e r t i l i z a t i o n , and the s i t e was able to meet these requirements. R e f e r r i n g to Table 13 i n the f i r s t year on s i t e 4, the c o n t r o l f o l i a r Zn c o n c e n t r a t i o n was 8.7 ug g - 1 , and was i n c r e a s e d to 17.2 ug g - 1 i n response to the "complete-Zn-Mn" treatment. F o l l o w i n g the second year i t was 11.7 ug g - 1 and the t r e a t e d l e v e l was 11.1 ug g - x • On s i t e 5 i n the f i r s t year the c o n t r o l f o l i a r Zn c o n c e n t r a t i o n was 11.4 ug g ~ x and i n c r e a s e d to 15.5 jig g~x with the "complete-Zn-Mn" treatment. In the second year the c o n t r o l l e v e l was 11.8 pg g - 1 and the treatment i n c r e a s e d f o l i a r Zn to 14.7 pg g _ x . I t i s i n t e r e s t i n g to see how the p a t t e r n of Zn response f o l l o w s the p a t t e r n of n i t r o g e n response. They both occurred only i n the f i r s t year on s i t e 4 and i n years one and two on s i t e 5. -There was not a great d i f f e r e n c e i n f o l i a r N l e v e l s f o r the second year c o n t r o l s on s i t e s 4 and 5, 1.17 and 1.15 cg g ~ x r e s p e c t i v e l y , yet there was a r e s i d u a l N response on s i t e 5. 2. Shoot Increment Ratio For a l l the s o i l Zn a p p l i c a t i o n s on a l l the s i t e s , a growth response occurred only on s i t e 2 to treatment 2 (50 kg Zn h a - t ) i n the second year (Table 14). 104 T a b l e 1 4 . S h o o t i n c r e m e n t r a t i o g r o w t h r e s p o n s e t o z i n c . V a l u e s i n p a r e n t h e s e a r e t h e s t a n d a r d d e v i a t i o n . An (*) i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e and n . s . d . i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e a t t h e 5% l e v e l b e t w e e n a t r e a t m e n t a n d i t s r e s p e c t i v e s u l f u r c o n t r o l . S i t e Tear Treatment Shoot Increment Ratio 2 2 SO kg Zn ha" 1 1.58 (0.53) 25 kg S ha" 1 1.14 (0.53) * 1 2 3600 mg Zn L" 1 1.61 (0.71) 1770 mg S L - 1 0.87 (0.23) * 4 2 360 mg Zn L~ l 1.86 (1.22) 177 mg S L" 1 1.14 (0 .39) 1800 mg Zn L" 1 1.75 (0.70) 883 mg S L" 1 1.46 (0.20) 2700 rag Zn L - 1 1.43 (0.32) 1325 mg S L" 1 1.30 (0.34) n.s.d. 5 2 360 mg Zn L _ 1 177 mg S L~ l 1800 mg Zn L 883 mg S L - 1 2700 mg Zn L" 1 1325 mg S L" 1 1.91 (0.89) 1.20 (0.29) 1.60 (0.54) 1.38 (0.41) n.s.d. 1.77 (0.43) 1.29 (0.54) 3 1 360 mg Zn L - 1 177 mg S L- 1 3600 mg Zn L ~ l 1770 mg S L- 1 2 360 mg Zn L" 1 177 mg S L - 1 1.18 (0.20) 0.86 (0.27) * 0.78 (0.38) 0.82 (0.23) n.s.d. 1.02 (0.49) 0.86 (0.37) n.s.d. 3600 mg Zn L" 1 1.02 (0.32) 1770 mg S L - 1 0.79 (0.30) 105 P o s i t i v e growth response to f o l i a r Zn a p p l i c a t i o n s occurred only i n the second year. On s i t e 1, i t took place at the highest a p p l i c a t i o n r a t e (treatment 5 (3600 mg Zn L _ t ) ) (Table 14). On s i t e 4 i t was detected with treatments 13 (360 mg Zn L~ l) and 14 (1800 mg Zn L~M (Table 14), and on s i t e 5 with treatments 13 (360 mg Zn L - t ) and 15 (2700 mg Zn L"M(Table 14). On s i t e 3 i t occurred i n both years but to d i f f e r e n t l e v e l s of Zn (Table 14). In the f i r s t year, response took p l a c e to treatment 4 (360 mg Zn L _M, and i n the second year to treatment 5 (3600 mg Zn L~M. P o s i t i v e growth response to s o i l - a p p l i e d Mn occurred on s i t e s 4 and 5 i n the f i r s t year (Table 15). On s i t e 4 response occurred to a l l Mn treatments. On s i t e 5, growth response was s i g n i f i c a n t o n l y to the 400 kg ha - 1- treatment. P o s i t v e growth response occurred to the "complete-Zn-Mn" treatment on both s i t e s 4 and 5 (Table 16) i n the f i r s t year, and i n the second year on s i t e 5. 106 Table 15. Shoot increment r a t i o response to s o i l a p p l i c a t i o n s of manganese i n the f i r s t year (1986) on s i t e s 4 and 5. An (*) i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e and n.s.d i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e at the 5X l e v e l between a treatment and i t s r e s p e c t i v e s u l f u r c o n t r o l . S i t e 4 S i t e 5 Treatment Shoot Increment Ratio - 200 kg Mn h a " 1 1.03 (0.26) 1.25 (0.35) 118 kg S h a " 1 0.83 (0.27) 1.09 (0.35) * n.s.d. 400 kg Mn h a " 1 1.15 (0.38) 1.21 (0.52) 235 kg S h a " 1 0.85 (0.28) 0.98 (0.37) * * 600 kg Mn h a " 1 1.18 (0.37) 1.23 (0.39) 352 kg S h a " 1 0.90 (0.32) 1.08 (0.39) * n.s.d. Table 16. Shoot increment r a t i o response to the "complete-Zn-Mn" treatment. An (*) i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e and n.s.d i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e at the 5% l e v e l between a treatment and i t s r e s p e c t i v e s u l f u r c o n t r o l . S i t e Year Treatment Shoot Increment Ratio 4 1 Complete-Zn-Mn 1.23 (0.48) C o n t r o l 0.76 (0.22) * 5 1 Complete-Zn-Mn 1.58 (0.67) C o n t r o l 0.96 (0.27) * 5 2 Complete-Zn-Mn 1.94 (1.11) C o n t r o l 1.39 (0.14) 108 3. Height Increment Where growth response to Zn i n terms of height growth increment o c c u r r e d , i t was not evident u n t i l i n the second year (Table 17). On s i t e 1 there was a s i g n i f i c a n t response to treatment 5 (3600 mg Zn L " M , and a trend to response on s i t e 2. On s i t e S, the response occurred from treatment 13 (360 mg Zn L - M , and on s i t e 4 there was a trend towards a response. In the f i r s t year of treatment f o r these s i t e s the Zn treatments tended to depress growth. Height increment response to f o l i a r Mn treatments d i d not occur u n t i l i n the second year (Table 17). T h i s was found on s i t e 5 f o r treatment 17 (4095 mg Mn L ~ * ) . On s i t e 5 response to s o i l Mn treatments occurred i n the f i r s t year to treatments 4 (200 kg Mn ha-*) and 6 (600 kg Mn h a " 1 ) . On s i t e s 4 and 5 i n the second year there was a trend towards a response to treatments 4 (200 kg Mn h a - 1 ) and 5 (600 kg Mn ha" 1) r e s p e c t i v e l y . C. R e t r a n s l o c a t i o n Z i n c and manganese c o n c e n t r a t i o n s were compared s e p a r a t e l y between f i r s t year f o l i a g e from 1985 and 1986, and between one- y e a r - o l d and two-year-old f o l i a g e from s i t e s 1 and 2 (Appendix M). For f o l i a r Zn from the low treatment (treatment 4) there was a decrease i n the two-year-old f o l i a g e but l e v e l s i n the f o l i a g e formed i n the second year were not d i f f e r e n t from the c o n t r o l 109 T a b l e 1 7 . H e i g h t i n c r e m e n t r a t i o r e s p o n s e . An (*) i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e and n . s . d . i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e a t t h e 5% l e v e l b e t w e e n a t r e a t m e n t a n d i t s r e s p e c t i v e s u l f u r c o n t r o l . Site Tear Treatnent Height Increment Ratio I 2 360 mg Zn l " 1 1.15 (0.61) 117 mg S L" 1 0.97 (0.73) n.s.d. 3600 mg Zn L" 1 2.54 (1.14) 1770 ng S L - l 0.83 (0.30) 2 2 360 mg Zn L - 1 1.57 (0.85) 117 mg S L" 1 1.42 (0.41) n.s.d. 3600 mg Zn L" 1 1.85 (1.62) 1770 mg S L " l 1.57 (0.20) n.s.d. 4 2 360 »g Zn L" 1 3 .30 (3 , .02) 117 mg S L" 1 2 n . 35 .s.d , (1 , .66) 5 2 360 mg Zn L- 1 2 .36 (1 . 28) 117 M S L - 1 1 .43 * (0. ,46) 200 kg Mn ha" 1 1 . .82 (0. , 76 ) 118 kg S ha" 1 1 , .24 * (0 , . 46 ) 400 kg Mn ha" 1 1 , .52 (0. .71) 235 kg S ha" 1 1 . , 69 (0 . 87) n . s.d. 600 kg Mn ha" 1 1 . 74 (0. .53) 352 kg Mn ha" 1 1 , .27 * (0. .57) 4095 mg Mn L" 1 2 .22 (1 . 49) 2408 mg Mn L~ 1 I .26 (0 . 67) 110 (Table 18). The f o l i a r Zn l e v e l s f o r the high treatment (treatment 5) decreased f o r the two-year-old f o l i a g e , and were e l e v a t e d f o r the f o l i a g e formed i n the second year r e l a t i v e to the c o n t r o l (Table 19). The i n c r e a s e d Zn c o n c e n t r a t i o n i n the f o l i a g e formed i n the second year and the decrease i n the two- y e a r - o l d f o l i a g e suggests t h a t there was r e t r a n s l o c a t i o n i n the second year, of Zn from the o l d to the new f o l i a g e . T h i s depended upon the l e v e l of the f o l i a r Zn treatment, with r e m o b i l i z a t i o n o c c u r r i n g f o r the high f o l i a r Zn treatment. On s i t e 1 the Zn . r e t r a n s l o c a t i o n was a s s o c i a t e d with a growth response i n terms of shoot and height increment r a t i o (Tables 14 and 17, r e s p e c t i v e l y ) . Manganese l e v e l s were higher i n the two-year-old f o l i a g e than the one-year-old f o l i a g e (Table Z0). The f o l i a g e formed i n the second year a l s o had e l e v a t e d l e v e l s of Mn r e l a t i v e to the c o n t r o l (Table 20). Both these r e s u l t s i n d i c a t e that the two- y e a r - o l d f o l i a g e , as w e l l as the c u r r e n t year's f o l i a g e formed i n the second season f o l l o w i n g f e r t i l i z a t i o n , continued to take up Mn. This suggests that i n c r e a s e d uptake w i l l occur i f s o i l Mn i s a v a i l a b l e . In the study of n u t r i e n t r e t r a n s l o c a t i o n i t ' was assummed that there was no change i n u n i t f o l i a r mass with f o l i a g e age. T h e r e f o r e , f o l i a r n u t r i e n t c o n c e n t r a t i o n s would no be changed from the d i l u t i o n or c o n c e n t r a t i o n s e f f e c t s . There i s evidence which i n d i c a t e s t h a t the decrease i n f o l i a r n u t r i e n t I l l Table 18. Change i n f o l i a r z i n c (ug g~M of c u r r e n t year's f o l i a g e over time and with age of f o l i a g e f o l l o w i n g treatment f o r the low z i n c f o l i a r treatment on s i t e s 1 and 2. Treatment 4 i s 360 mg Zn L~x and treatment 7 i s 177 mg S L~x. S i t e 1 Time Year s i n c e treatment Treatment Zn 4 C o n t r o l 7 t - t e s t S i t e 2 1 2 t - t e s t 51.2 13.6 * 5.1 11.5 * * n.s.d Age Age s i n c e treatment 51.2 5.1 * 2 48.7 19.8 * t - t e s t n.s.d * Time Year s i n c e treatment Treatment 1 2 t - t e s t Zn 4 53.0 11.5 * C o n t r o l 7 2.0 8.8 n.s.d t - t e s t * n.s.d Age Age s i n c e treatment 53.0 2.0 * 2 35 .2 15.6 * t - t e s t * n.s.d 112 Table 19. Change i n f o l i a r z i n c (pg g~M of c u r r e n t year's f o l i a g e with time and with age of f o l i a g e f o l l o w i n g treatment f o r the high z i n c f o l i a r treatment on s i t e s 1 and 2. Treatment 5 i s 3600 mg Zn L - 1 and treatment 8 i s 1770 mg S L - t . S i t e 1 Time Age Years s i n c e treatment Age s i n c e treatment Treatment 1 2 t - t e s t 1 2 t - t e s t Zn 5 144.4 15.4 * 144.4 135.5 * C o n t r o l 8 2.5 11.0 * 2.5 17.9 * t - t e s t * * * * S i t e 2 Time Age Years s i n c e treatment Age s i n c e treatment Treatment 1 2 t - t e s t 1 2 t - t e s t Zn 5 139.6 19.6 * 139.6 78.7 * C o n t r o l 8 2.5 11.4 * 2.5 17.0 * t - t e s t * * * * 113 Table 20. Change i n C o l l a r manganese (pg g~M of c u r r e n t year's f o l i a g e with time and with age of f o l i a g e f o l l o w i n g treatment f o r the manganese s o i l treatment (3) on s i t e s 1 and 2. Treatment 3 i s 200 kg Mn h a - 1 and treatment 12 i s the untreated c o n t r o l . S i t e 1 Time Years s i n c e treatment Treatment 1 2 t - t e s t Mn 3 3059 2971 n.s.d C o n t r o l 12 1019 1139 n.s.d t - t e s t * * Age Age s i n c e treatment 1 3059 1019 * 2 4610 1432 * t - t e s t * S i t e 2 Time Years s i n c e treatment Treatment 1 2 t - t e s t 1 Mn 3 3196 3875 * 3196 C o n t r o l 12 1474 1932 * 1474 Age Age s i n c e treatment 2 5920 2365 t - t e s t * * t - t e s t I 114 c o n c e n t r a t i o n s i n the two-year-old f o l i a g e was not due to f o l i a r l e a c h i n g . With an i n c r e a s e i n f o l i a g e age, f o l i a r Zn c o n c e n t r a t i o n s f o r the c o n t r o l s i n c r e a s e d (Tables 18 and 19) i n d i c a t i n g no evidence of Zn l e a c h i n g . D. Nutrient-Growth I n t e r a c t i o n s Data were f i t t e d to the model y = a + bx + c x 2 , where y i s a measure of growth and x i s f o l i a r Zn c o n c e n t r a t i o n , to t e s t the hypothesis that the r e l a t i o n s h i p of f o l i a r Zn c o n c e n t r a t i o n to growth f o l l o w s the model of d i m i n i s h i n g r e t u r n s . S i g n i f i c a n t r e g r e s s i o n s were found between height increment and f o l i a r Zn c o n c e n t r a t i o n s (Figure 22 and Table 21). These were found between c u r r e n t year's height increment and f o l i a r Zn f o r 1986 and 1987 f o r s i t e s 4 and 5. Comparing the f o l i a r l e v e l s of the optimum height increment to the c o n t r o l Zn l e v e l s suggests that height increment b e n e f i t e d from the higher f o l i a r Zn c o n c e n t r a t i o n s . There were no r e l a t i o n s h i p s found between other growth parameters e i t h e r measured or c a l c u l a t e d and f o l i a r n u t r i e n t c o n c e n t r a t i o n s . 115 F i g u r e 2 2 . S c a t t e r p l o t s o f h e i g h t i n c r e m e n t (cm) v e r s u s c u r r e n t y e a r ' s f o l i a r z i n c l e v e l s ( p g g - 1 ) . ( A ) I n t h e f i r s t y e a r ( 1 9 8 6 ) ( f o r c a s e s w h e r e Zn <50 pg g - 1 ) and ( B ) i n t h e s e c o n d y e a r ( 1 9 8 7 ) f o r s i t e 4 . 116 Table 21. Equations and c o r r e l a t i o n c o e f f i c i e n t s f o r the height increment (y) - f o l i a r z i n c (x) r e l a t i o n s h i p s f o r s i t e s 4 and 5. Refer to Appendix N f o r s c a t t e r p l o t s other than given i n Figure 22. S i t e Year Equation R a 4 1986 y • 5.125x - 0.094x a 0.81 4 1987 y = -31.677 + 15.209 - 0.481x a 0.37 5 1986 y = 2.709x - 0.03x a 0.22 5 1987 y = 8.577x - 0.234x a 0.18 117 E. N u t r i e n t I n t e r a c t i o n s 1. I n t e r a c t i o n s with Z i n c a. Manganese On s i t e 5 i n the f i r s t year, treatment 15 (2700 mg Zn L~M produced the onl y i n c i d e n t where Zn a p p l i c a t i o n reduced f o l i a r Mn (Table 22). Treatment 1 (50 kg Mn h a - 1 ) a c t u a l l y i n c r e a s e d f o l i a r Mn. b. Nitrogen S c a t t e r p l o t s of f o l i a r N versus f o l i a r Zn r e v e a l e d s i g n i f i c a n t r e l a t i o n s h i p s between the two (F i g u r e 23 and Table 23). In a d d i t i o n , a l i n e a r r e l a t i o n s h i p was found to e x i s t between e x t r a c t a b l e s o i l Zn and t o t a l s o i l N c o n c e n t r a t i o n s (Figure 24). 118 Table 22. F o l i a r manganese (pg g"*) n u t r i e n t c o n c e n t r a t i o n response to s o i l and f o l i a r a p p l i c a t i o n s of z i n c i n the f i r s t , year (1986) on s i t e 5. S o i l Treatments F o l i a r Treatments Treatment Mn Treatment Mn 50 kg Zn h a " 1 1262 Q37) 25 kg S ha~* 1178 (416) 100 kg Zn ha"* 1273 (209) 49 kg S ha"* 1525 (670) n.s.d. 200 kg Zn ha-* 1289 (548) 98 kg S ha~* 1327 (536) n.s.d. 360 mg Zn L~* 1217 (235) 117 mg S L-* 1140 (249) n.s.d. 1800 mg Zn L~* 1066 (319) 883 mg S L~* 1272 (751) n.s.d. 2700 mg Zn L~* 867 (487) 1325 mg S L~* 1094 (261) * 119 1.60 -i OO 1.20 - CT> C CU ?0 .80 H 0.40 - y = 0 . 5 2 + 0 . 0 7 6 x - 0 . 0 0 1 6 2 x : R a = 0 . 5 2 (B) 0.00 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 0.0 10.0 20.0 30.0 Foliar Zinc 1986 1 1 1 1 1 40.0 F i g u r e 2 3 . S c a t t e r p l o t s o f f o l i a r n i t r o g e n ( c g g - 1 ) v e r s u s f o l i a r z i n c ( p g g - 1 ) o f t h e c u r r e n t y e a r ' s f o l i a g e ( A ) i n t h e f i r s t y e a r ( 1 9 8 5 ) ( f o r c a s e s w h e r e Z n <50 pg g - 1 ) and ( B ) i n 1986 ( f o r c a s e s w h e r e Zn <40 pg g - 1 ) f r o m s i t e 1 . The c o r r e l a t i o n c o e f f i c i e n t ( R 2 ) i s g i v e n . 120 Table 23. Equations and c o r r e l a t i o n c o e f f i c i e n t s (R a) f o r f o l i a r n i t r o g e n (y) - f o l i a r z i n c (x) r e l a t i o n s h i p s a c cording to s i t e . Refer to Appendix 0 f o r s c a t t e r p l o t s other than given i n Figure 23. S i t e Year Equation R 2 1 1985 y • 0.52 + 0.07x - 0.00162x a 0.52 1 1986 y • 0.6 + 0.047x - 0.0009x a 0.21 2 1985 y = 0.6 + 0.0997x - 0.0033tx a 0.44 2 1986 y = 0.8 + 0.038x - 0.0009x a 0.21 4 1986 y = 0.l37x - 0.0036lx a 0.46 4 1987 y - 0.55 + 0.074x - 0.00134x a 0.50 5 1986 y = 0.306 + 0.0932x - 0.00l76x a 0.65 5 1987 y • 0.234 + 0.115x - 0.00293x a 0.54 121 F i g u r e 2 4 . S c a t t e r p l o t o f t o t a l s o i l n i t r o g e n ( e g g - 1 ) v e s u s e x t r a c t a b l e s o i l z i n c (ug g - 1 - ) b o t h f r o m t h e f o r e s t f l o o r f o r a l l s i t e s . 122 2. I n t e r a c t i o n s with Manganese a. Z i n c Manganese f o l i a r and s o i l treatments a f f e c t e d f o l i a r Zn l e v e l s on s i t e 1 (Table 24). F o l i a r - a p p l i e d Mn i n c r e a s e d f o l i a r Zn i n both the f i r s t and second y e a r s . The Mn s o i l treatment (200 kg Mn ha~M i n c r e a s e d f o l i a r Zn i n the f i r s t year. On s i t e 5, f o l i a r Zn was a l s o i n c r e a s e d by s o i l a p p l i c a t i o n s of Mn i n the f i r s t year from treatments 4 (200 kg Mn ha~M and 6 (600 kg Mn h a - 1 ) (Table 25). As shown i n Figure 25 t h i s was a synergism of f o l i a r Zn to Mn a p p l i c a t i o n s ( s h i f t i n C d i r e c t i o n ) . There were no other r e l a t i o n s h i p s found between Mn treatments and f o l i a r c o n c e n t r a t i o n s of other n u t r i e n t s . T h e r e f o r e , i t i s u n l i k e l y t h at the growth responses obtained with Mn s o i l treatments were due to the a l l e v i a t i o n of t o x i c i t i e s of other n u t r i e n t s or m e t a l l i c elements. Table 24. F o l i a r z i n c (ug g " M n u t r i e n t c o n c e n t r a t i o n response to s o i l and f o l i a r manganese a p p l i c a t i o n s on s i t e 1. F o l i a r Treatment Treatment 1985 1986 Zn Zn u g g-l. 2730 mg Mn L " v 9.4 (2.5) 10.6 (1.5) C o n t r o l 7.2 (1.7) 8.8 (2.3) * * S o i l Treatment Treatment 1985 Zn -ug g ~ x - 200 kg Mn h a " 1 15.1 (5.8) C o n t r o l 7.6 (1.9) Table 25. F o l i a r z i n c (ug g - 1 ) n u t r i e n t response i n the f i r s t year (1986) to s o i l a p p l i c a t i o n s of manganese on s i t e 5. S o i l Treatments Treatment 200 kg Mn h a " 1 118 kg S h a " 1 400 kg Mn h a ' 1 235 kg S h a " 1 600 kg Mn h a - 1 352 kg S h a " 1 Zn -ug g - 1 — 11.9 (2.3) 8.3 (1.5) * 12.1 (5.2) 10.5 (2.7) n.s.d. 15.7 (5.8) 10.1 (1.4) 125 F i g u r e 2 5 . V e c t o r d i a g r a m o f f i r s t y e a r g r o w t h r e s p o n s e ( i n 1 9 8 6 ) o f Zn t o s o i l t r e a t m e n t s o f Mn f r o m s i t e 5 . The x - a x i s i s ug Z n p e r s h o o t , t h e y - a x i s i s ug Z n g - 1 a n d t h e d a s h e d l i n e i s f o l i a r mass p e r s h o o t i n g r a m s . The a r r o w i n d i c a t e s t h e d i r e c t i o n o f t h e r e s p o n s e . T r e a t m e n t s a r e i n k g Mn h a - 1 . 126 F. R e l a t i o n s h i p of Response to S i t e A summary of n u t r i e n t and growth responses to f o l i a r and s o i l Zn treatments are presented i n Tables 26 and 27 r e s p e c t i v e l y . There was no obvious r e l a t i o n s h i p between response and s i t e c o n d i t i o n s . In s i m i l a r manner, f o r the s o i l Mn treatments (Table 28) there was no r e l a t i o n s h i p between response and s i t e . An i n t e r e s t i n g r e l a t i o n s h i p was found between s i t e c o n d i t i o n s and response to the "complete-Zn-Mn" treatment- on s i t e s 4 and 5 (Table 29). There are d i s t i n c t d i f f e r e n c e s between s i t e s 4 and 5. S i t e 4 i s CWHbl, low e l e v a t i o n and has a humo- f e r r i c podzol with an Ae h o r i z o n . In c o n t r a s t s i t e 5 i s CWHb2, high e l e v a t i o n and has a ferro-humic podzol with an enriched o r g a n i c B h o r i z o n . There was a response to N, Zn and B i n the f i r s t year r e g a r d l e s s of s i t e ( F i g u r e s 20 and 21A). On s i t e 5 response to N and the Zn synergism continued i n t o the second year (F i g u r e 21B). In a d d i t i o n there were responses to Cu and Fe i n the f i r s t year (Figure 21A) and a strong response to P i n the second year (Figure 21B). 127 Table 26. Summary of n u t r i e n t and growth responses to f o l i a r Zn treatments. A (*) i n d i c a t e s a s i g n i f i c a n t i n c r e a s e , a (**) i n d i c a t e s a s i g n i f i c a n t decrease and a (-) i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e at the 52 l e v e l between a treatment and i t s c o n t r o l . F i r s t Year Second Year S i t e Treatment Zn Mass Shoot Height Zn Mass Shoot Heigh (mg Zn L - l ) Ratio Ratio Ratio Ratio 1 360 * 3600 * — — — * — * * 2 360 * * — - - — - - 3600 * ** - — * — — — 3 360 * - * * - -3600 — — — — * — 4 360 * - - - - - * -1800 * - - - - - * -2700 * — - - - — — — 5 360 * ** — — _ — * — 1800 * - - - - - - * 2700 * - - - - - * - 128 Table 27. Summary of n u t r i e n t and growth responses to s o i l Zn treatments. An (*) i n d i c a t e s a s i g n i f i c a n t i n c r e a s e and a (-) i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e at the 5X l e v e l between a treatment and i t s c o n t r o l . Second Year S i t e Treatment Zn Hass Shoot Height (kg Zn ha-1) Ratio Ratio 10 * 50 * 10 50 10 50 * 50 100 200 * 50 100 200 * 129 Table 28. Summary of n u t r i e n t and growth responses to s o i l Zn treatments. An (*) i n d i c a t e s a s i g n i f i c a n t i n c r e a s e , a (**) i n d i c a t e s a s i g n i f i c a n t decrease and a (-) i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e at the 5% l e v e l between a treatment and i t s c o n t r o l . F i r s t Year Second Year S i t e Treatment Mn Mass Shoot Height Mn Mass Shoot Height (kg Mn ha-l) Ratio Ratio Ratio Ratio 1 200 * - - - * - - • 2 200 * - - - * - _ 3 200 - - * - - 4 200 — — * — * ** — — 400 * - * - * - -600 * — * - * * — — 5 200 * * — * * _ - — 400 * * * - * - -600 * * - * * - - 130 T a b l e 2 9 . Summary o f n u t r i e n t and g r o w t h r e s p o n s e s t o t h e " c o m p l e t e - Z n - M n " t r e a t m e n t . An (*) i n d i c a t e s a s i g n i f i c a n t i n c r e a s e and a ( - ) i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e a t t h e 5% l e v e l b e t w e e n a t r e a t m e n t and i t s c o n t r o l . F i r s t Y e a r S i t e N P Zn Fe A c t i v e Fe Cu B M a s s S h o o t H e i g h t R a t i o R a t i o 4 * _ * _ _ - * * * 5 * — * * * * * * * P S e c o n d Y e a r Zn Mass S h o o t R a t i o H e i g h t R a t i o 4 * 5 * * 131 CHAPTER 5. DISCUSSION A. COMPARATIVE NUTRITION 1. T o t a l L e v e l s Hemlock had lower f o l i a r Zn c o n c e n t r a t i o n s and higher f o l i a r Mn c o n c e n t r a t i o n s compared to D o u g l a s - f i r , a m a b i l i s f i r and red cedar. Z a s o s k i er al. (1990) a l s o r e p o r t e d f o l i a r Zn c o n c e n t r a t i o n s to be lower and f o l i a r Mn c o n c e n t r a t i o n s to be higher than some other c o n i f e r s i n the P a c i f i c Northwest. The f a c t that the comparison i n t h i s study was made among s p e c i e s on the same s i t e s and i n the same stands would i n d i c a t e t h a t the l e v e l s of Mn and Zn i n hemlock are p l a n t - r e l a t e d and not s o i l - r e l a t e d . In a d d i t i o n , i t i s u n l i k e l y d i f f e r e n t l e v e l s of f o l i a r Zn and Mn between s p e c i e s are due to the e x p l o i t a t i o n of d i f f e r e n t s o i l h o r i z o n s by the root systems. Root excavations done by E i s (1987) found that on s i m i l a r s o i l s , r o o t s of Douglas- f i r , cedar and hemlock t r e e s penetrated to s i m i l a r depth and t r e e s of s i m i l a r s i z e extended t h e i r root system over s i m i l a r areas. Since 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 r o o t i n g depth between s p e c i e s of 50-year-old t r e e s , i t may be safe to assume that the r o o t i n g depth of s p e c i e s of younger t r e e s , as used i n t h i s study, would not be s i g n i f i c a n t l y d i f f e r e n t . Even i f hemlock was e x p l o i t i n g the f o r e s t f l o o r more than other s p e c i e s , t h i s part of the s o i l h o r i z o n has the highest a v a i l a b l e Zn (Table 30). T a b l e 3 0 . S o i l d a t a f o r a l l s i t e s . ' T * r e f e r s t o t h e t o p h o r i z o n w h i c h was f o r e s t f l o o r and ' B ' r e f e r s t o t h e t o p 15 cm o f t h e m i n e r a l s o i l h o r i z o n . MEAN SITE ZnT ZnB Mnl MnB KT KB AIT A l B MS g - 1 1 22.1 2.9 49 .6 5 . 1 196 .1 55 . 4 1116 1707 3 15.2 1.8 226.0 13. 6 431 .0 188 .6 1037 1758 4 29.2 5.8 44.4 21. 0 198 .4 166 .0 874 1495 5 23 . 7 1.8 47.6 2 . 8 249 .6 28 .6 1176 1762 STANDARD DEVIATION 1 17.6 1.6 40 .2 6 . 5 121 .4 41 .8 438 289 3 6.7 1.3 51.3 3. 1 190 .5 45 .2 364 343 4 10.6 3.6 21.8 35 . 9 48 .1 95 .5 325 210 5 13.4 0.7 27.6 2 . 3 204 .0 14 .5 425 541 MEAN SITE CaT CaB MgT MgB PT FB CuT CuB b 1 1 879 80 208 23 . ,2 56 . 3 12 . 7 0.96 0.40 3 1175 210 350 48 . , 7 58. 0 6. 9 0 .64 0.56 4 1447 233 267 64. .0 70 . 3 17 . 5 1 .32 0 .82 5 1268 137 288 22 . 1 72 . 6 7 . 2 1.22 0 .96 STANDARD DEVIATION 1 688 77 178 21. .2 53 . 3 16 . 1 0.59 0 .26 3 416 113 17 20 . 6 26 . 7 5 . 8 0 .17 0 .16 4 541 200 145 56 . 2 25 . 4 18 . 1 0 . 22 0.50 5 706 68 141 6 . 9 48 . 8 7 . 8 0.50 0 .63 BT BB EXNT EXTNB TNT TNB pHT pHB g ~ l eg g - 1 H a0 0.47 0. 16 0 .016 0 . 004 0 . 60 0 .19 3 . 8 4 , . 1 0.40 0. 11 0 .063 0 .016 0 .67 0 .24 4 .5 4, .6 0.03 0 . 19 0 .013 0 .005 0 .90 0 . 24 4 .0 3 . 8 0 . 14 0 . 05 0 .017 0 .005 0 .93 0 .20 4 . 1 4 , .5 0 . 78 0 . 06 0 , .021 0 . 002 0 , .38 0 . 10 0 , .3 0 . ,3 0.03 0 . 04 0 . 044 0. .010 0 , .07 0 , .11 0 . 3 0 . 3 0.01 0 . 11 0 , .012 0 , .001 0 . 26 0 . 14 0 . 6 0 . , 7 0.09 0 . 06 0 , .020 .0 , .003 0 . 24 0 , .11 0 .3 0 . 4 FeT FeB pHT pHB CECT CECB --Cf i C l a ' — -meq lOOg" 184 226 3 .5 3 .8 12 .9 14.2 261 285 3 .9 4 .1 6 . 7 19 .6 123 217 3 .4 3 . 7 7 . 7 13.2 179 177 3 .5 3 . 7 9 . 4 12.4 43 118 0 .4 0 . 4 6 . 1 2.8 37 252 0 . 1 0 .4 1 .2 7 . 4 38 114 0 . 4 0 .2 2 .8 . 6.9 89 93 0 . 3 0 .5 3 . 1 6 . 4 U) 133 The d i f f e r e n c e s among s p e c i e s may be a t t r i b u t e d to d i f f e r e n c e s i n the r e g u l a t i o n of n u t r i e n t uptake and/or d i f f e r e n c e s i n the r e g u l a t i o n of n u t r i e n t t r a n s p o r t between root and shoot. Among the p o s s i b l e reasons f o r unusual n u t r i e n t c o n c e n t r a t i o n s of Zn and Mn may be d i f f e r e n t p h y s i o l o g i c a l requirements and/or e c o l o g i c a l s t r a t e g i e s i n hemlock. A s i t u a t i o n s i m i l a r to hemlock has been found f o r black spruce and jack p i n e . Black spruce has higher f o l i a r c o n c e n t r a t i o n s of manganese as compared with jack pine (Morrison and Armson 1968; Lafond and Laflamme 1968). T h i s i s an example of how the p h y s i o l o g y of the p l a n t i s t i e d i n t o i t s ecology. Morrison and Armson (1968) contend that the high Mn l e v e l s of the spruce f o l i a g e c r e a t e Mn-rich s u r f a c e l a y e r s which allow r e g e n e r a t i o n of spruce but i n h i b i t the r e g e n e r a t i o n of jack p i n e . 2. E x t r a c t a b l e Zn and Mn D o u g l a s - f i r has higher water-soluble Zn than hemlock but am a b i l i s f i r has s i m i l a r l e v e l s of water-soluble Zn to hemlock. T h i s may suggest that D o u g l a s - f i r has a higher component of a c t i v e z i n c , which might i n d i c a t e a higher requirement f o r Zn than i n hemlock and a m a b i l i s f i r . The higher t o t a l and water- s o l u b l e Zn i n white pine than hemlock which i n turn had a higher l e v e l than cedar suggests a r e l a t i v e p h y s i o l o g i c a l requirement i n the order pine>hemlock>cedar. 134 The h i g h e r l e v e l o f w a t e r - s o l u b l e Mn i n h e m l o c k t h a n D o u g l a s - f i r , a m a b i l i s f i r , p i n e and c e d a r may i n d i c a t e a h i g h e r r e q u i r e m e n t f o r Mn b y h e m l o c k . 3 . C e l l u l a r F r a c t i o n s o f F o l i a g e F r a c t i o n s o f f o l i a g e r e p r e s e n t d i f f e r e n t c e l l u l a r c o m p o n e n t s w h i c h a r e i n v o l v e d i n s p e c i f i c p h y s i o l o g i c a l p r o c e s s e s . F o r e x a m p l e , f r a c t i o n B and C r e p r e s e n t t h e c h l o r o p l a s t i c c o m p o n e n t w h i c h a r e i n v o l v e d i n t h e p r o c e s s o f p h o t o s y n t h e s i s . H e m l o c k had l o w e r Zn i n m o s t f r a c t i o n s c o m p a r e d t o D o u g l a s - f i r . T h i s r e l a t i o n s h i p i s c o n s i s t e n t w i t h t o t a l f o l i a r l e v e l s . T h e r e f o r e t o t a l f o l i a r Z n c o n c e n t r a t i o n s may r e f l e c t p h y s i o l o g i c a l l e v e l s o f Z n . The m e t h o d u s e d t o s e p a r a t e f r a c t i o n E i s s i m i l a r t o t h e m e t h o d u s e d b y R a h i m i and S c h r o p p ( 1 9 8 4 ) , i n w h i c h t h e y m e a s u r e d t h e p h y s i o l o g i c a l l y a c t i v e Zn a s s o c i a t e d w i t h c a r b o n i c a n h y d r a s e a c t i v i t y . T h e r e f o r e t h e Zn l e v e l i n t h e E f r a c t i o n may be t h e p h y s i o l o g i c a l l y a c t i v e Z n . H e m l o c k had h i g h e r Mn l e v e l s c o m p a r e d t o o t h e r s p e c i e s i n a l l f r a c t i o n s . T h e s e r e s u l t s f o r Mn may have s e v e r a l i n t e r p r e t a t i o n s . H i g h e r l e v e l s may mean t h a t h e m l o c k h a s a g r e a t e r p h y s i o l o g i c a l r e q u i r e m e n t f o r M n , o r a l t e r n a t i v e l y may r e p r e s e n t some Mn a c c u m u l a t i o n as a t o l e r a n c e m e c h a n i s m . I T h e r e f o r e , i n d i c a t i o n t o t a l f o l i a r Mn l e v e l s may n o t o f p h y s i o l o g i c a l l y a c t i v e M n . n e c e s s a r i l y be an 135 The o n l y o t h e r s t u d y w h i c h h a s e x a m i n e d Mn i n c e l l u l a r f r a c t i o n s o f f o l i a g e was by Memon ( 1 9 8 4 ) . He e x a m i n e d t h e m a n g a n e s e c o n c e n t r a t i o n o f d i f f e r e n t c e l l f r a c t i o n s i n t h e f o l i a g e o f a Mn a c c u m u l a t o r . M a n g a n e s e was h i g h e s t i n t h e s u p e r n a t a n t f r a c t i o n w h i c h i n t h i s s t u d y was p a r t o f f r a c t i o n E . B . F e r t i l i z a t i o n E x p e r i m e n t s 1 . N u t r i e n t and G r o w t h R e s p o n s e s T h e r e w e r e p o s i t i v e l i n e a r and c u r v i l i n e a r r e l a t i o n s h i p s b e t w e e n c u r r e n t h e i g h t i n c r e m e n t and f o l i a r mass p e r s h o o t . T h e r e i s e v i d e n c e w h i c h s u p p o r t s a r e l a t i o n s h i p b e t w e e n f o l i a r mass and g r o w t h . F o r e x a m p l e , t h e r e i s a c u r v i l i n e a r r e l a t i o n s h i p b e t w e e n l e a f a r e a i n d e x and t h e n e t p r i m a r y p r o d u c t i v i t y . N e t p r i m a r y p r o d u c t i v i t y r e a c h e s a maximum w i t h a s p e c i f i c l e a f a r e a i n d e x b u t a b o v e t h i s p o i n t y i e l d i s r e d u c e d b e c a u s e o f h i g h r e s p i r a t o r y l o s s e s r e q u i r e d t o m a i n t a i n a l a r g e v o l u m e o f l e a f and s u p p o r t i n g t i s s u e s ( K r a m e r and K o z l o w s k i 1 9 7 9 ) . F o r d ( 1 9 8 4 ) r e v i e w s a s t u d y i n w h i c h a p o s i t i v e c o r r e l a t i o n was f o u n d b e t w e e n a n n u a l b r a n c h - a n d - b o l e p r o d u c t i o n 136 and f o l i a g e biomass f o r both c o n i f e r s and deciduous t r e e s . However, the study d i d not show as the authors c l a i m that there i s "no decrease i n dry matter production even at the highest l e a f biomass". To do t h i s they should have demonstrated no evidence of a c u r v i l i n e a r r e l a t i o n s h i p (Ford 1984). Th e r e f o r e , i t has not been proven that t r e e growth may continue to i n c r e a s e i n d e f i n i t e l y as f o l i a g e amount i n c r e a s e s (Ford 1984). Some equations f i t t e d to data from t h i s t h e s i s suggest that there may be a decrease; however, unambiguous evidence has not been obtained. a. Zinc i . Comparison of F o l i a r Versus S o i l Treatments The f o l i a r and s o i l methods of a p p l i c a t i o n were used i n order to t e s t whether some f a c t o r ( s ) [e.g. of the s o i l , the uptake mechanism, or t r a n s l o c a t i o n ] was i n t e r f e r i n g with the movement of s o i l - a p p l i e d z i n c to the f o l i a g e . The f o l i a r hemlock n u t r i e n t data i n d i c a t e the g r e a t e r e f f i c i e n c y of Zn uptake with f o l i a r a p p l i c a t i o n s compared to s o i l a p p l i c a t i o n s . F o l i a r a p p l i c a t i o n s of Zn have been found to be up to 12 times more e f f i c i e n t than s o i l a p p l i c a t i o n s (Murphy and Walsh 1972). Stout er al. (1987), found that with a l f a l f a , Zn recovery i n the p l a n t was l e s s than 2% of the s o i l - a p p l i e d Zn. Since i n hemlock there was a delay i n z i n c response to s o i l a p p l i c a t i o n s , and the changes i n f o l i a r c o n c e n t r a t i o n were not as dramatic as the 137 f o l i a r treatments, one or more of the above mentioned f a c t o r s may have accounted f o r the p a t t e r n of response observed f o r s o i l Zn a p p l i c a t i o n s . In a g r i c u l t u r e , z i n c i s a p p l i e d to the s o i l f o r row crops and as a f o l i a r spray f o r t r e e and vine crops (Traynor 1980). Chevis (1983) found that a 2.5 mg L - x z i n c sulphate f o l i a r spray r a i s e d the f o l i a r z i n c c o n c e n t r a t i o n s i n Pinus radiata, c o r r e c t i n g a d e f i c i e n c y , while s o i l a p p l i c a t i o n s of z i n c oxide di d not c o r r e c t the d e f i c i e n c y . S e e d l i n g s of P. e l l i o t t i i grown i n pots took up z i n c i n the f o l i a g e when z i n c was a p p l i e d to the s o i l i n a s o l u t i o n form (van Lear and Smith 1972). McKee (1976) found that s e e d l i n g s of P. e l l i o t t i i responded to z i n c a p p l i e d i n s o l u t i o n form to the s o i l i n a pot experiment. S e e d l i n g s of P. radiata d i d not d i s p l a y symptoms of z i n c d e f i c i e n c y when i t was s u p p l i e d i n s o l u t i o n c u l t u r e (Smith and B a y l i s s 1942). McGrath (1978) concluded that a p p l i c a t i o n of z i n c as a f o l i a r spray was a more e f f e c t i v e and r e l i a b l e method of s u p p l y i n g z i n c to P. radiata than a d d i t i o n to the s o i l of z i n c oxide with superphosphate. Symptoms of z i n c d e f i r . i « n c y w « r e o v « r r ; o m e when a IX s o l u t i o n of z i n c c h l o r i d e was a p p l i e d to the f o l i a g e ( K e s s e l and Stoate 1936). F o l i a r a p p l i c a t i o n s of 0.6 mg L~x z i n c to s e e d l i n g s of P. radiata i n the nursery r a i s e d t h e i r f o l i a r z i n c c o n c e n t r a t i o n s and prevented v i s u a l symptoms of z i n c d e f i c i e n c y (Knight 1975). A f o l i a r a p p l i c a t i o n of 9,800 ug g - i of z i n c c h e l a t e c o r r e c t e d the growth d i s o r d e r s of 46-month-old t r e e s of P. caribaea (Sance et al. 1982). Stoate (1950) found that f o l i a r 138 a p p l i c a t i o n s of 2.5 mg L - t z i n c sulphate as a f o l i a r spray or s o i l d r e s s i n g s of 0.12 grams to 1.8 kg z i n c sulphate to i n d i v i d u a l t r e e s prevented the c o n t i n u a t i o n of growth d i s o r d e r s i n P. radiata. F i r s t l y , a v a i l a b i l i t y may have been a f f e c t e d by f a c t o r s of the s o i l . A v a i l a b i l i t y of Zn from the s o i l may be a problem due to t r a n s p o r t from the s o i l to the r o o t . Autoradiographs of wheat root s using s s Z n have i n d i c a t e d zones of d e p l e t i o n around the r o o t s . T h i s d e p l e t i o n i n d i c a t e s the c r e a t i o n of a c o n c e n t r a t i o n g r a d i e n t , and that the i o n moves i n part by d i f f u s i o n (Wilkinson er al. 1968). Since d i f f u s i o n i s important i n the t r a n s p o r t of the i o n , root contact with the s o i l i s important i n the a b s o r p t i o n of z i n c (Boawn et al. 1957). This i s demonstrated by the f a c t t h a t a v a i l a b i l i t y of z i n c i s i n c r e a s e d when i t i s mixed with the s o i l r a t h e r than a p p l i e d i n a band (Shaw er al. 1954; Murphy and Walsh 1972). Wider d i s t r i b u t i o n would i n c r e a s e i t s contact with r o o t s (Murphy and Walsh 1972). The s o i l pH a f f e c t s the a v a i l a b i l i t y of z i n c through i t s s o l u b i l i t y . S t u d i e s examining the i n f l u e n c e of pH on z i n c a d s o r p t i o n show a decrease i n z i n c s o l u b i l i t y with i n c r e a s i n g pH (Saed and Fox 1977; Bar-Yosef 1979; McBride and B l a s i a k 1979). The s o l u b i l i t y of z i n c decreases 100-fold f o r each u n i t i n c r e a s e i n pH ( T i s d a l e er al. 1985). With s o i l a c i d i f i c a t i o n , p l a n t uptake of s o i l Zn or banded a p p l i c a t i o n s were i n c r e a s e d ( V i e t s er al. 1957). 139 In a study by Maclean (1974), corn, l e t t u c e and a l f a l f a p l a n t s were grown with Zn l e v e l s s i m i l a r to those found i n the s o i l s used i n t h i s study (Table 30). I t i s i n t e r e s t i n g to note that the s o i l s had a DTPA-extractable Zn of 26.8 pg g - 1 , and a pH of 4.9 while the corresponding f o l i a r l e v e l s of the p l a n t s were 238 ( c o r n ) , 523 ( l e t t u c e ) , and 321 ( a l f a l f a ) pg g - 1 , which were t o x i c to growth. Since the s o i l Zn l e v e l s found by Maclean (1974) were t o x i c to p l a n t s , Zn a v a i l a b i l i t y i n hemlock may be l i m i t e d by uptake or some i n t e r a c t i o n but not by s o i l supply. Two a d d i t i o n a l sources of i n f o r m a t i o n suggest t h i s . F i r s t , some other c o n i f e r s p e c i e s on the same s i t e s have higher f o l i a r Zn c o n c e n t r a t i o n s than hemlock. Second, i n c r e a s e d uptake of Zn occurred with the "complete-Zn-Mn" treatment i n d i c a t i n g a d d i t i o n a l s o i l Zn was a v a i l a b l e . T h erefore, with the s o i l pH l e v e l s of the study s i t e s being r e l a t i v e l y low (3-4), the high i n h e r e n t supply of a v a i l a b l e Zn, the use of broadcast a p p l i c a t i o n s , and the use of the s o l u b l e ZnSO*, i t i s u n l i k e l y t h a t there was a problem of Zn f i x a t i o n , i n s o l u b i l i t y or low s o i l Zn l e v e l s . The second p o s s i b l e reason i n v o l v e s the uptake and t r a n s l o c a t i o n of z i n c . T h i s i n v o l v e s two aspects; f i r s t l y the p h y s i o l o g i c a l requirements of the p l a n t f o r Zn, and secondly the e c o l o g i c a l aspect of metal t o l e r a n c e . Hemlock may accumulate Zn i n the r o o t s and r e s t r i c t t r a n s p o r t from the r o o t s to the shoots, 140 or r e s t r i c t uptake from the s o i l to the r o o t . Work done with s e v e r a l s p e c i e s [soybean (White er al. 1979), bush beans (Ruano et al. 1988; Hawf and Smith 1967), maize and b a r l e y (Singh and Steenberg 1974), maize (Nair and Prabhat 1977), v a r i o u s s p e c i e s ( C a r r o l l and Loneragan 1968), and subterranean c l o v e r (Riceman and Jones 1958)] i n d i c a t e s t hat with Zn a p p l i c a t i o n , Zn accumulates i n the roo t s r e l a t i v e to the shoot. The delay i n f o l i a r response to s o i l a p p l i c a t i o n s i s s i m i l a r to the s i t u a t i o n found by West (1979) with Cu i n P. radiata. Roots tended to accumulate Cu, but over time there was a net decrease i n root Cu l e v e l s and an i n c r e a s e i n shoot Cu l e v e l s . I t appears that some of the excess Cu, s t o r e d i n roo t s which had been exposed to high Cu l e v e l s , was r e l e a s e d to shoots when s u f f i c i e n t Cu was no longer a v a i l a b l e (West 1979). The r e s u l t s of the f o l i a r and s o i l Zn treatments suggest that i t i s f a c t o r s of the p l a n t r e l a t e d to uptake and/or t r a n s l o c a t i o n which account f o r the observed p a t t e r n of response to Zn, and hence the c h a r a c t e r i s t i c Zn n u t r i t i o n of hemlock. i i . Z i n c Tolerance Of the three types of p l a n t - s o i l n u t r i t i o n a l r e l a t i o n s h i p s (accumulator, excluder and i n d i c a t o r ) , hemlock may be an excluder with r e s p e c t to Zn, judging from the p a t t e r n of response to s o i l Zn treatments. Observations of m e t a l - r e s i s t a n t p l a n t s 141 from d i f f e r e n t e c o l o g i c a l s i t u a t i o n s show that t o l e r a n c e i s not achieved by e x c l u s i o n with r e s p e c t to uptake (Ernst,1975). R e s t r i c t i o n of t r a n s p o r t from root to shoot i s the l i k e l y mechanism of c o n t r o l (Baker 1981; Woolhouse 1983; Foy er al. 1978). Consequently, metal c o n c e n t r a t i o n s i n the shoot are maintained constant and low over a wide range of s o i l c o n c e n t r a t i o n s , up to a c r i t i c a l s o i l value above which the mechanism breaks down and u n r e s t r i c t e d t r a n s p o r t r e s u l t s (Baker 1981). This phenomenon of Zn e x c l u s i o n has been examined i n s e v e r a l s p e c i e s . For example, d i f f e r e n t p o p u l a t i o n s of Silene maritima from Zn-contaminated s o i l s , when grown i n s o l u t i o n c u l t u r e , excluded Zn from t h e i r shoots (Thurman 1981). Agrostis tenuis i s a s p e c i e s of grass which has z i n c - t o l e r a n t clones i n which t o l e r a n c e i s c o n f e r r e d on the p l a n t through i n c r e a s e d r e t e n t i o n of Zn i n the r o o t s (Woolhouse 1983). Z i n c - t o l e r a n t clones of the grasses Deschampsia caespitosa and Anthoxanthum odoratum concentrated Zn i n t h e i r r o o t s (Brookes er al. 1981). Seedlings of hemlock grown i n s o l u t i o n c u l t u r e were found to have higher z i n c c o n c e n t r a t i o n s i n the r o o t s compared to the f o l i a g e (Table 31) (Zasoski er al. 1990). In e x c l u d e r s , d e t o x i f i c a t i o n occurs l a r g e l y w i t h i n the r o o t s (Baker 1981). The mechanisms by which Zn may be d e t o x i f i e d i n the root are i m m o b i l i z a t i o n of Zn i n c e l l w a l l s and compartmentation as a s o l u b l e complex, fre e i o n or i n s o l u b l e complex ( m e t a l l o t h i o n e i n ) (Woolhouse 1983). 142 Table 31. E f f e c t of s o l u t i o n pH on m i c r o n u t r l e n t c o n c e n t r a t i o n s (mg k g - 1 ) i n D o u g l a s - f i r and western hemlock r o o t s (R) and needles (L) (from Zasoski er al. 1990)). S o l u t i o n pH 3 3 .5 4 4.5 5 Douglas - f i r Fe R 424 666 1180 1810 2240 L 78 80 90 95 88 Mn R 21 27 25 27 30 L 87 97 100 102 97 Zn R 90 87 78 80 74 L 47 45 46 48 43 Cu R 53 84 95 129 144 L 28 32 30 34 31 Western Hemlock Fe R 301 779 1950 3290 3780 L 104 112 116 141 142 Mn R 34 37 37 39 42 L 265 2 70 266 289 256 Zn R 78 69 74 84 77 L 35 41 33 39 34 Cu R 54 63 78 110 117 L 54 55 60 66 63 143 Denny and W i l k i n s (1987a), s t u d y i n g z i n c t o l e r a n c e i n Betula, r e j e c t e d the t o l e r a n c e mechanisms of i n t e r n a l d e t o x i f i c a t i o n and c e l l w all b i n d i n g . ' They found using m i c r o a n a l y s i s that at Zn c o n c e n t r a t i o n s above the l e t h a l l e v e l , Zn accumulated i n t r a c e l l u l a r l y , at the endodermis, i n the form of electron-dense g r a n u l e s . They a l s o i n v e s t i g a t e d the mechanism of e c t o m y c o r r h i z a l a m e l i o r a t i o n of z i n c t o x i c i t y to Betula. As the fungal hyphae penetrate the s o i l , Zn i s adsorbed to the s u r f a c e of hyphae, thereby lowering the c o n c e n t r a t i o n of z i n c i n the s o i l s o l u t i o n surrounding the r o o t s . The metal may a l s o be adsorbed to e l e c t r o - n e g a t i v e s i t e s i n the hyphal c e l l w a l l s and to e x t r a - hyphal, p o l y s a c c h a r i d e slime (Denny and W i l k i n s 1987b). Another method of d e t o x i f i c a t i o n has been i n v e s t i g a t e d by van Steveninck er al. (1987). They found high l e v e l s of Zn i n g l o b u l e s i n the r o o t s of z i n c - t o l e r a n t ecotype of Deschampsia caespitosa. The g l o b u l e s appear to occur i n small vacuoles w i t h i n the cytoplasmic matrix of e l o n g a t i n g c e l l s i n the c o r t e x . An a d d i t i o n a l mechanism i s that Zn may be concentrated by the mycorrhizae. Zinc has been found to be concentrated i n the r o o t s of m y c o r r h l z a l p l a n t s of Pinus virginiana ( M i l l e r and Rudolph 1986), Betula (Brown and W i l k i n s 1985), and Ericaceous p l a n t s (Bradley er al. 1982) compared to u n i n f e c t e d p l a n t s . Since hemlock r o o t s are known to be h i g h l y m y c o r r h l z a l ( G i l l and Lavender 1983), i t i s p o s s i b l e t h a t such a mechanism i s o p e r a t i n g i n hemlock. b. Manganese 144 i . Comparison oE F o l i a r Versus S o i l Treatments N u t r i e n t responses to s o i l a p p l i c a t i o n s of Mn were much more e f f i c i e n t than f o l i a r a p p l i c a t i o n s . In a d d i t i o n , responses were c o n s i s t e n t with s o i l a p p l i c a t i o n s . The reasons for the d i f f e r e n t responses between Zn and Mn when a p p l i e d as a f o l i a r spray r e q u i r e an examination of the process of s o l u t e movement through the f o l i a g e as o u t l i n e d i n Figure 26. F o l i a r uptake of n u t r i e n t s i n v o l v e s d e p o s i t i o n , a b s o r p t i o n through the c u t i c l e , epidermal c e l l s (Bukovac and Wittwar 1957), and the stomates ( S w i e t l i k and Faust 1984), ad s o r p t i o n on the s u r f a c e of the plasma membranes, passage through the plasma membranes and movement i n t o the cytoplasm ( S w i e t l i k and Faust 1984). I n i t i a l p e n e t r a t i o n of the c u t i c l e occurs by d i f f u s i o n , which depends upon temperature and a c o n c e n t r a t i o n g r a d i e n t ( S w i e t l i k and Faust 1984). Manganese d e f i c i e n c y was c o r r e c t e d i n P. radiata using f o l i a r sprays with Mn at 8.5 and 8.75 mg L " 1 compared to the f o l i a r l e v e l s of 10 pg g~ x ( R u i t e r 1983). The c o n c e n t r a t i o n of manganese i n the s o l u t i o n may have been too small r e l a t i v e to the c o n c e n t r a t i o n i n the f o l i a g e f o r d i f f u s i o n to occur. In a g r i c u l t u r e , f o l i a r a p p l i c a t i o n of Mn i s one of the most e f f i c i e n t ways of c o r r e c t i n g Mn d e f i c i e n c y (Murphy and Walsh 1972), and MnS0«, which was used i n t h i s study, i s considered to be the most e f f e c t i v e i n o r g a n i c c a r r i e r of Mn (Murphy and Walsh 1972). 145 Surface wetting , Cutfcular penetration Active transport across the plasma lemma ApopJastic free apace movement Synthesis of organic compounds Sympiastic movement via the piasmadesmata Energy-dependent phloem loading Energy-dependent phloem loading Translocation F i g u r e 2 6 . P o s s i b l e p a t h w a y s o f s o l u t e movement t h r o u g h t h e l e a f ( f r o m H a y n e s ( 1 9 8 6 ) ) . 146 Therefore the la c k of response to f o l i a r a p p l i e d Hn found i n t h i s t h e s i s i s probably not due to the a p p l i c a t i o n method per se or the type of c a r r i e r . D i f f e r e n c e s i n the f o l i a r uptake and t r a n s l o c a t i o n of Zn and Mn have been demonstrated i n s e v e r a l s t u d i e s . Bukovac and Wittwar (1957) found that 6 B Z n moved through the t r a n s p i r a t i o n stream, whereas Mn tended to concentrate where i t was a p p l i e d and a g r e a t e r percentage of the Zn was absorbed. Romey and Toth (1954) found B*Mn to be absorbed through the f o l i a g e although there were d i f f e r e n c e s between p l a n t s p e c i e s i n the amount absorbed. But, Hn was again found to concentrate i n the i n t e r v e i n a l t i s s u e s , forming small i s l a n d s . Chamel (1985) found Mn to be p o o r l y absorbed, s i n c e d i s c s which were i n i t i a l l y charged with s*Mn l o s t almost a l l the a p p l i e d Mn by washing, whereas 80% of the e s Z n was l o s t . He concluded that c u t i c u l a r r e t e n t i o n i s dependent upon the element c o n s i d e r e d . Manganese has been found to be r e a d i l y leached from f o l i a g e (Tukey er al. 1958). However, i n another study with pea p l a n t s there was 100X ab s o r p t i o n of Mn i n t o the t r e a t e d area of the l e a f (Ferrandon and Chamel 1988). In t h i s study, the c h a r a c t e r i s t i c s of the element, the c o n c e n t r a t i o n of the s o l u t i o n , and the c h a r a c t e r i s t i c s of the f o l i a g e may have a l l c o n t r i b u t e d to the low Mn a b s o r p t i o n by the hemlock f o l i a g e . i i . T o x i c i t y L e v e l s 147 The r e s u l t s I n d i c a t e a growth response to s o i l - a p p l i e d Mn. Th i s i s i n t e r e s t i n g because the f o l i a r l e v e l s f o r the c o n t r o l t r e e s are higher than t o x i c i t y l e v e l s r e p o r t e d f o r agronomic crop p l a n t s . Reported t o x i c i t y l e v e l s are 1,000 ug g _ 1 i n beans, 550 pg g _ 1 i n peas, and 200 pg g - i i n b a r l e y (White 1970), 380 pg g~x i n Medicago s a t i v a , 1,600 pg g ~ x i n Centrosena pubescens, > 2,600 pg g - x i n c a r r o t s , > 160 pg g - x i n Bragg soybeans, 450-500 pg g - x i n tomato (young l e a v e s ) (Foy er al. 1978), 120-600 pg g - x i n apple, 445-1400 pg g - t , r e s p e c t i v e l y , i n upper and lower leaves of sweet sorghum, 500 pg g - x i n f l a x , 500-1,960 pg g - x i n c o t t o n , 2,000 pg g ~ x i n Easter l i l y , 2,600 pg g - x i n c a r n a t i o n , 2,500- 6,500 pg g - x i n maize (Foy 1983), 1,200-2,600 pg g - x i n c o f f e e , and 120 pg g"1- i n snap beans (Foy 1984). There are s e v e r a l examples of Mn f o l i a r l e v e l s and the occurrence of t o x i c i t y symptoms i n f o r e s t t r e e s . Dying European f i r s e e d l i n g s having blackened r o o t s suggestive of a Mn t o x i c i t y had f o l i a r l e v e l s of 1,300 to 2,500 pg g - x compared to 120 to 500 f o r l i v i n g s e e d l i n g s (Stone 1967). A 60% r e d u c t i o n i n growth of black spruce and jack pine s e e d l i n g s grown i n s o l u t i o n c u l t u r e was noted when the c u l t u r e s o l u t i o n contained 100 pg g - x Mn, and the corresponding f o l i a r Mn l e v e l s of the s e e d l i n g s were 4,200 to 4,400 pg g - x (Morrison and Armson 1966). Although hemlock had higher or s i m i l a r f o l i a r Mn l e v e l s f o r the Mn s o i l treatments compared to the t o x i c i t y l e v e l s r e p o r t e d i n the l i t e r a t u r e f o r other p l a n t s , there was no evidence of a Mn t o x i c i t y i n t h i s study. 148 i i i . Manganese Requirements The f o l i a r Mn l e v e l s are higher f o r hemlock than f o r other t r e e s p e c i e s i n the same stands. In a d d i t i o n , there was a f o l i a r n u t r i e n t c o n c e n t r a t i o n response to s o i l a p p l i c a t i o n s of Mn, without a r e d u c t i o n i n growth. These f a c t s suggest that hemlock i s manganese-tolerant and has a gr e a t e r t o t a l Mn requirement compared to some other B.C. c o n i f e r s . T h i s i s c o n s i s t e n t with other s t u d i e s which have repo r t e d an i n c r e a s e d need f o r metals i n m e t a l - t o l e r a n t ecotypes of many s p e c i e s . Examples of these are wheat (Mn) (Macfie 1989; Foy er al. 1973), copper moss (Scopelopbila cataractae) (Shaw 1987), Agrostis tenuis (Cu, Pb, Zn), Mimulus guttatus (Cu), Anthoxanthum odoratum (Zn), ffolcus lanatus (Zn), Armeria maritima (Zn), Silene vulgaris (Zn) (Antonovics 1971), Succisa pratensis (Mn) (Pe g t e l 1986), Avenella Flexuosa, Chamaenerion angustifolium, Rumex acetosella, and Senecio sylvaticus (Mn) (Ernst and N e l i s s e n 1979). T h i s phenomenon has been ex p l a i n e d by in v o k i n g z i n c - t o l e r a n t p l a n t s . If a t o l e r a n t p l a n t complexes the metal i n order to d e t o x i f y i t , one would expect a shortage i n these p l a n t s on normal s o i l s ( E rnst 1975). The biomass p r o d u c t i o n would be diminished and can be s t i m u l a t e d by Zn c o n c e n t r a t i o n s which are al r e a d y t o x i c f o r no n - t o l e r a n t p l a n t s . The a c t i v i t y of c a r b o n i c anhydrase was found to be s t i m u l a t e d at high amounts of z i n c i n t o l e r a n t p o p u l a t i o n s , but not i n n o n - t o l e r a n t p o p u l a t i o n s (Ernst 1975). The r e f o r e , because of the e f f i c i e n c y of the t o l e r a n c e mechanism 149 i n i n a c t i v a t i n g the metals, the e x t e r n a l t r a c e element requirement i s higher (Antonovics er al. 1971). v i . Manganese Tolerance Hemlock i s s i m i l a r to other p l a n t s growing on a c i d i c s u b s t r a t e s i n that i t i s a manganese accumulator which enables i t to t o l e r a t e t h i s type of environment. These types of p l a n t s are c l a s s i f i e d as c a l c i f u g e s , which are p l a n t s adapted to low s o i l pH. Ericaceae i s a f a m i l y of p l a n t s which l i k e hemlock are c a l c i f u g e s . Species i n the Ericaceae belonging to the genus Vaccinium a l s o occur i n stands of hemlock. T h i s i s of i n t e r e s t because some s p e c i e s of Vaccinium are of agronomic importance and work has been done on t h e i r n u t r i t i o n . Of the three types of p l a n t - s o i l r e l a t i o n s h i p s , hemlock may be c l a s s i f i e d as an accumulator with r e s p e c t to Mn. T h i s i s s i m i l a r to the Vaccinium where i n t e r n a l t o l e r a n c e e s p e c i a l l y i n l e a f t i s s u e i s the mechanism of t o l e r a n c e (Korcak 1988). F o l i a r l e v e l s of Mn i n Vaccinium s p e c i e s have been repo r t e d i n excess of 2,000 to 4,000 pg g - 1 (Korcak 1988). There are s e v e r a l mechanisms by which the high l e v e l s of manganese i n the f o l i a g e may be d e t o x i f i e d . One hypothesis i s that of compartmentalization (Foy er al. 1978). This i s where ions are excluded from the a c t i v e p a r t s of the c e l l and concentrated i n vacuolar and other i n e r t r e g i o n s as complexes or ions (Ernst 1975). There i s f u r t h e r support f o r compartmentation as a 150 d e t o x i f y i n g mechanism. Memon and Yatazawa (1984) i d e n t i f i e d a manganese-oxalate complex i n the supernatant f r a c t i o n of l e a f c e l l s of the manganese accumulator Acanthopanax sciadophylloides. The supernatant c o n s t i t u t e d the vacuolar f r a c t i o n and had the high e s t Mn c o n c e n t r a t i o n of a l l the f r a c t i o n s . S i m i l a r l y , hemlock may t o l e r a t e high Mn by i s o l a t i n g i t i n vacuoles s i n c e , from the c e l l u l a r f r a c t i o n study, t h i s f r a c t i o n had the highest Mn l e v e l . The Mn may be i n the fr e e i o n i c s t a t e s i n c e most of the f o l i a r Mn was wate r - s o l u b l e , or i t may be weakly complexed judging from Memon and Yatazawa's f i n d i n g s (1982, 1984). C. Comparison and C o n s i d e r a t i o n of Adequate L e v e l s . i . I n d i v i d u a l N u t r i e n t s According to vecto r a n a l y s i s some of these hemlock stands had d e f i c i e n c i e s of n u t r i e n t s i n a d d i t i o n to Zn and Mn (Fi g u r e s 20 and 21). Although vect o r a n a l y s i s i n d i c a t e s a stro n g Zn d e f i c i e n c y there were strong responses to other n u t r i e n t s . It i s i n t e r e s t i n g to co n s i d e r these r e s u l t s i n l i g h t of the laws of l i m i t i n g f a c t o r s . There are two laws which d e s c r i b e the r e l a t i o n of growth to l i m i t i n g f a c t o r s . L i e b i g ' s law of the minimum suggests growth i s c o n t r o l l e d by the most l i m i t i n g f a c t o r . That i s growth response to a n u t r i e n t w i l l not occur unless d e f i c i e n c y of the most l i m i t i n g n u t r e i n t i s at f i r s t a l l e v i a t e d . T h i s law i s analogous to a b a r r e l with staves of d i f f e r e n t s i z e s . The a b i l i t y of the b a r r e l to hold water i s l i m i t e d by the s h o r t e s t 151 s t a v e . T h i s law may be l i m i t e d to the s i t u a t i o n i n b i o l o g y where the minimum f a c t o r i s so low as to stop the process e n t i r e l y ( D a n i e l er al. 1979), perhaps where v i s u a l symptoms of a d e f i c i e n c y are e v i d e n t . M i t s c h e r l i c h ' s law of the minimum argues that i n c r e a s i n g any f a c t o r that i s below i t s optimum w i l l improve growth, but i n c r e a s i n g the f a c t o r f u r t h e s t from i t s optimum w i l l give the g r e a t e s t i n c r e a s e i n growth (Daniel et al. 1979). A response occurred to a "complete-Zn-Mn" treatment even though Zn was shown from vector a n a l y s i s as being s t r o n g l y l i m i t i n g . This treatment was s y n e r g i s t i c to Zn uptake. T h e r e f o r e , i t i s d i f f i c u l t to say whether response was due to a n u t r i e n t i n the "complete-Zn-Mn" treatment, the Zn, or an improvement i n n u t r i e n t balance. According to the " b a r r e l " analogy, a growth response should not have occurred i n t h i s study unless the Zn d e f i c i e n c y was f i r s t a l l e v i a t e d . T h i s seems to demonstrate the l i m i t e d a p p l i c a b i l i t y of the " b a r r e l " approach i n b i o l o g i c a l systems. I t does not recognize that there are i n t e r a c t i o n s between n u t r i e n t s , such as synergisms or antagonisms, or a n u t r i e n t may improve growth c r e a t i n g a s i n k f o r another n u t r i e n t . The response data from the "complete-Zn-Mn" treatment (Table 13) were compared to e x i s t i n g f o l i a r n u t r i e n t i n t e r p r e t a t i o n s (Appendix I ) , to assess i f the i n t e r p r e t a t i o n s from the g u i d e l i n e s were c o n s i s t e n t with the observed r e s u l t s . B a l l a r d and C a r t e r (1985) i n t h e i r review (Appendix I) i n d i c a t e an adequate f o l i a r N t h r e s h o l d of 1.45 eg g - 1 . Both 152 s i t e s had f o l i a r V l e v e l s below 1.0 cg g""1 and 1.11 cg g - 1 i n the year of f e r t i l i z a t i o n and they responded to 100 kg ha~ x of N. T h i s i s i n the s e v e r e l y d e f i c i e n t range. F e r t i l i z a t i o n brought f o l i a r l e v e l s up to 1.67 cg g - t f o r both s i t e s , which i s adequate. Hardie (1985) found that 100 kg h a - 1 of N and 150 kg h a - 1 of P d i d not a f f e c t f o l i a r N c o n c e n t r a t i o n s but t o t a l s e e d l i n g dry mass was i n c r e a s e d over the c o n t r o l . The c o n t r o l and t r e a t e d f o l i a r c o n c e n t r a t i o n s were both above the adequate t h r e s h o l d . Weetman et al. (1989) have a l s o i d e n t i f i e d d e f i c i e n c i e s of N and P i n young r e g e n e r a t i o n of c o a s t a l western hemlock using f o l i a r a n a l y s i s . D e f i c i e n c i e s of N and P were confirmed by subsequent 3-year height growth response. Data from G i l l and Lavender (1983) showed a response to 224 kg N h a - 1 , with c o n t r o l t r e e s having f o l i a r N l e v e l s of 1.33 to 1.53 cg g ~ x . Seedlings i n the f i e l d having f o l i a r N l e v e l s of 0.96 cg g - 1 ( s e v e r e l y d e f i c i e n t ) responded to 200, 300 and 400 kg N h a - 1 (Germain 1984). Zasoski and Gessel (1982) found that s e e d l i n g s having a f o l i a r N l e v e l of 0.88 cg g ~ x (very s e v e r e l y d e f i c i e n t ) responded to a p p l i c a t i o n of 198 kg N h a - 1 . In a study of 940 p l o t s of immature stands of D o u g l a s - f i r and hemlock i n c o a s t a l B.C. measured over 12 years, 50 - 70% of hemlock stands responded to N f e r t i l i z a t i o n . F e r t i l i z a t i o n * i n c r e a s e d both t o t a l and merchantable volume but height was not s i g n i f i c a n t l y a f f e c t e d . Unthinned hemlock stands on f r e s h and moist s i t e s responded to f e r t i l i z a t i o n b e t t e r than d i d those on the moderately dry s i t e s (Omule 1990). 153 A f t e r the f i r s t year, s i t e 4 had a c o n t r o l P l e v e l of 0.197 eg g - * and s i t e 5 had 0.182 eg g - 1 . A f t e r the second year, s i t e 4 had a f o l i a r P l e v e l of 0.183 eg g - 1- and s i t e 5 had 0.167 eg g - 1 . When f o l i a r P dropped to 0.167 eg g - x on s i t e 5 a f t e r the second year, there was a n u t r i e n t response to P. When the f o l i a r P l e v e l was higher there was no response. There have been r e p o r t s of hemlock response to P f e r t i l i z a t i o n . Hardie (1985) found i n c r e a s e d P i n s e e d l i n g s f e r t i l i z e d with 150 kg P h a - 1 with c o n t r o l s having a mean f o l i a r P l e v e l of 0.124 eg g _ 1 . B a l l a r d and C a r t e r (1985) suggest 0.15-0.35 eg g"~* r e p r e s e n t s the range of s l i g h t l y d e f i c i e n t to adequate. Data from Heilman and Ekuan (1980) i n d i c a t e that a P response to 300 kg of P ha-*- occurred to s e e d l i n g s with f o l i a r P l e v e l s i n the s e v e r e l y d e f i c i e n t range (<0.11 eg g ~ M . Germain (1984) found that a p p l i c a t i o n s of P at e i t h e r 50 kg h a - 1 or 38 kg h a - 1 i n c r e a s e d f o l i a r P l e v e l s of s e e d l i n g s i n the f i e l d which had c o n t r o l l e v e l s of 0.13 pg g - 1 . Zasoski and Gessel (1982) found that s e e d l i n g s which had a f o l i a r l e v e l of 0.08 eg g _ 1 , which i s considered d e f i c i e n t , responded to a P a p p l i c a t i o n of 447 kg h a - 1 . Adequate l e v e l s f o r f o l i a r B are suggested to be i n the range of 10-15 pg g""1 ( B a l l a r d and C a r t e r 1985). An experiment by Majid (1984) with greenhouse grown lodgepole pine s e e d l i n g s suggests a c r i t i c a l range f o r f o l i a r B of 7 to 16 pg g _ t . C o n t r o l f o l i a r B l e v e l s f o r s i t e s 4 and 5 were 24.5 and 28.4 pg g - 1- r e s p e c t i v e l y , and B f e r t i l i z a t i o n i n c r e a s e d them to 33.2 and 45.8 pg g ~ x r e s p e c t i v e l y . These are much g r e a t e r than the 154 suggested c r i t i c a l range and the B response v e c t o r s ( F i g u r e s 20 and 21) i n d i c a t e a g r e a t e r requirement f o r B. A f o l i a r c o n c e n t r a t i o n of 4 pg g _ l i s proposed to be an adequate l e v e l f o r Cu. F i e l d t r i a l s with lodgepole pine i n d i c a t e a c r i t i c a l value f o r f o l i a r Cu to be 4 pg g~ x (Majid 1984). F o l i a r Cu on s i t e 4 was 3.2 pg g - x and 3.1 f o r s i t e 5. F e r t i l i z a t i o n i n c r e a s e d f o l i a r Cu on s i t e 5 to 4 pg g - 1 which i s i n f e r r e d to be j u s t adequate with evidence of a d e f i c i e n c y from Figure 21A. Although Cu on s i t e 4 may be c l a s s i f i e d as p o s s i b l y inadequate there was no s i g n i f i c a n t response. F o l i a r S c o n c e n t r a t i o n s were 0.16 and 0.12 eg g - x for s i t e s 4 and 5 r e s p e c t i v e l y , and the N/S r a t i o of c o n t r o l f o l i a g e on s i t e 5 was 9.0. According to the d i a g n o s t i c g u i d e l i n e s i n Appendix I, s i t e 4 i s between a S d e f i c i e n c y u n l i k e l y and no S d e f i c i e n c y , there i s no S d e f i c i e n c y , and a N induced d e f i c i e n c y i s u n l i k e l y . The recommendations from the g u i d e l i n e s appear to be c o n s i s t e n t with the r e s u l t s of the S treatments i n which there was no i n c r e a s e d uptake, suggesting that S i s not l i m i t i n g . There was no response to Ca, K, or Mg. F o l i a r Ca was 0.28 and 0.32 eg g-*- f o r s i t e s 4 and 5, r e s p e c t i v e l y , which i s g r e a t e r than 0.2 eg g - t c r i t i c a l value which i s considered to be adequate. . F o l i a r K was 0.68 and 0.78 eg g~~x f o r s i t e s 4 and 5 r e s p e c t i v e l y . An adequate value f o r hemlock i s suggested to be 0.8 eg g ~ x . However, s i n c e there was no i n c r e a s e d n u t r i e n t 155 uptake, a lower value may perhaps be considered as adequate. F o l i a r Mg was 0.14 cg g-*- f o r both s i t e s , which was s u f f i c i e n t compared to the l e v e l considered adequate of 0.12 cg g - 1 . Since there was no response to Ca, K or Mg f e r t i l i z a t i o n , these l i m i t s c o n s idered adequate may be a p p l i c a b l e f o r hemlock. In the case of K t h i s l i m i t may be reduced. The i n c r e a s e i n f o l i a r a c t i v e Fe c o n c e n t r a t i o n on s i t e 5 a f t e r the f i r s t year suggests t h a t p h y s i o l o g i c a l l y a c t i v e i r o n i s l i m i t i n g . F o l i a r a c t i v e Fe went from 26.2 pg g - 1 f o r the c o n t r o l to 34.8 f o r the treatment. The g u i d e l i n e s suggest that 30 pg g - 1 separates the " d e f i c i e n c y l i k e l y " category from the " d e f i c i e n c y u n l i k e l y " category. T h i s seems to be a s u i t a b l e recommendation fo r hemlock s i n c e an Fe n u t r i e n t c o n c e n t r a t i o n response occurred when f o l i a r l e v e l s were below 30 pg g - 1 on s i t e 5, but there was no s i g n i f i c a n t response where f o l i a r l e v e l s where above 30 as on s i t e 4. F o l i a r Fe l e v e l s were 25 and 39 pg g - 1 f o r s i t e s 4 and 5 r e s p e c t i v e l y which are i n the range of p o s s i b l e d e f i c i e n c y (25-50 ug 8 _ 1>. C o n t r o l f o l i a r Zn c o n c e n t r a t i o n s (Table 8) are below the l e v e l considered to be adequate: 15 pg g~x . Since p o s i t i v e growth responses occurred to Zn f e r t i l i z a t i o n , the d i a g n o s t i c norms for Zn appear to adequately d e s c r i b e Zn n u t r i t i o n of hemlock. Further evidence of a Zn d e f i c i e n c y comes from the s t r o n g Zn response to the "complete-Zn-Mn" treatment ( F i g u r e s 20 and 21) 156 The d i a g n o s t i c norms for Mn need to be r e v i s e d f o r hemlock. Although f o l i a r Mn c o n c e n t r a t i o n s f o r the c o n t r o l s (Table 8) were higher than the " s t a t e d " adequate l e v e l of 25 pg g - 1 , growth response to Mn f e r t i l i z a t i o n o c c u r r e d . i i . N u t r i e n t Balance Ingestad's n u t r i e n t r a t i o s were c a l c u l a t e d f o r the f o l i a r n u t r i e n t s from the "complete -Zn -Mn" and the c o n t r o l treatments fo r s i t e s 4 and 5. These data are presented i n Table 32 along with Ingestad's r a t i o s considered to be optimum for growth (Ingestad 1979). The n u t r i e n t r a t i o s f o r both the "complete -Zn- Mn" and c o n t r o l treatments tend to be i n balance r e l a t i v e to the optimum r a t i o s . The exceptions are Mn and Fe which may be considered to be out of balance with respect to N, with Mn being too high and Fe being too low. T h i s may be an i n d i c a t i o n of an i n s u f f i c i e n t supply of Fe. The "complete -Zn -Mn" treatment tended to maintain the f o l i a r n u t r i e n t balance; however, a f t e r the f i r s t year of treatment, f e r t i l i z a t i o n tended to decrease the r a t i o s of K, P, and Cu from the optimum r a t i o s . Since there had been an i n c r e a s e i n f o l i a r P and Cu c o n c e n t r a t i o n s , the decrease to suboptimal r a t i o s suggests t h a t these l e v e l s are i n s u f f i c i e n t and f u r t h e r a d d i t i o n s are r e q u i r e d . 157 Table 32. Ingestad's f o l i a r n u t r i e n t r a t i o s f o r the complete -Zn -Mn and c o n t r o l treatments on s i t e s 4 and 5 f o r the years 1986 and 1987. The optimum r a t i o s are from Ingestad (1979). Zn Mn K Ca Mg P TS S i t e 4 1986 Complete 0.1 8 39.8 18.1 7.1 9.6 10.4 C o n t r o l 0.087 14.3 68 27.7 14 19.7 16 S i t e 4 1987 Complete 0.09 10.3 57.5 26.1 11.7 17 C o n t r o l 0.1 13.2 65.1 27.9 13.6 15.6 S i t e 5 1986 Complete 0.09 6.4 48.4 18.1 8.2 12.8 C o n t r o l 0.1 11.7 69.7 28.7 12.6 16.3 S i t e 5 1987 Complete 0.11 7.3 65.2 25.5 11.3 18.3 C o n t r o l 0.1 10.8 72.7 27.3 11.4 14.4 Table 28 (concluded). Fe Cu B AFe AZn ANn S i t e 4 1986 Complete 0.23 0.021 0.2 0.22 0.11 2.66 C o n t r o l 0.25 0.032 0.25 0.32 0.068 12.4 S i t e 4 1987 C o n t r o l 0.42 0.031 Complete 0.42 0.031 S i t e 5 1986 Complete 6.8 0.28 0.024 0.27 0.21 C o n t r o l 11.1 0.35 0.028 0.26 0.24 S i t e 5 1987 Complete 0.41 0.077 C o n t r o l 0.46 0.034 Ingestad's Ratio Zn Hn K Ca Mg P 0.03 0.4 70 8 5 16 TS Fe Cu B 9 7 0.03 0.2 159 Since there had been no i n c r e a s e i n the f o l i a r c o n c e n t r a t i o n of R but uptake had been maintained and there was a growth response, these r e s u l t s could suggest that the true optimum r a t i o f o r K i s a c t u a l l y not so high. D. R e t r a n s l o c a t i o n The e x i s t e n c e of n u t r i e n t d e f i c i e n c i e s may be assessed by determining the occurrence and the extent of n u t r i e n t r e m o b i l i z a t i o n . R e m o b i l i z a t i o n of mineral n u t r i e n t s i s important during ontogenesis i n per i o d s of i n s u f f i c i e n t supply of n u t r i e n t s to the r o o t s during v e g e t a t i v e growth (Kramer and Kozlowski 1979). If a pl a n t i s under a n u t r i e n t s t r e s s some n u t r i e n t s w i l l be r e m o b i l i z e d from the o l d f o l i a g e and t r a n s l o c a t e d to the new f o l i a g e . T h i s r e t r a n s l o c a t i o n could then be used as a means of diagnosing the n u t r i e n t s t a t u s of a p l a n t . In the second year f o l l o w i n g s o i l a p p l i c a t i o n s of Mn the cur r e n t year's f o l i a g e and two-year-old f o l i a g e continued to accumulate Mn. Ther e f o r e , i t could not be determined i f r e t r a n s l o c a t i o n o c c u r r e d . R e t r a n s l o c a t i o n of Zn occurred i n the second year f o l l o w i n g f e r t i l i z a t i o n from the the high f o l i a r Zn treatment. T h i s r e s u l t was i n c o n t r a s t to the s i t u a t i o n i n P. radiata found by McGrath and Robson (1984). In t h e i r s e e d l i n g s , the Zn c o n c e n t r a t i o n of the o l d e r primary needles remained constant r e g a r d l e s s of the Zn s t a t u s of the s e e d l i n g s . In c o n t r a s t , the r e s u l t s from t h i s r e s e a r c h f o r Zn are c o n s i s t e n t 160 with the f i n d i n g that r e m o b i l i z a t i o n of Zn from o l d leaves depends upon the Zn s t a t u s of the p l a n t . Zn i s more mobile when the c o n c e n t r a t i o n i s high (Kramer and Kozlowski 1979). For example, subterranean c l o v e r p l a n t s given l u x u r y s u p p l i e s of Zn moved up to 25% of the Zn i n t h e i r o l d leaves and p e t i o l e s i n t o d eveloping f r u i t s , whereas those given d e f i c i e n t or marginal s u p p l i e s moved l i t t l e or none at a l l (Loneragan 1976). There i s evidence that the decrease i n f o l i a r Zn c o n c e n t r a t i o n s i n the two-year-old f o l i a g e was due to r e m o b i l i z a t i o n . F i r s t l y , f o l i a r Zn c o n c e n t r a t i o n s i n the c o n t r o l f o l i a g e d i d not decrease with f o l i a r age. Secondly, Zn has been found to be leached with d i f f i c u l t y . Less than 1% of the Zn was leached with water a p p l i e d f o r 24 hours to young leaves of squash and beans (Tukey et al. 1958). E. N u t r i e n t I n t e r a c t i o n s A p p l i c a t i o n s of Zn had no e f f e c t on uptake of Mn and i n some cases a p p l i c a t i o n s of Hn i n c r e a s e d f o l i a r Zn. In soybeans, Zn has been shown to i n c r e a s e the t r a n s l o c a t i o n of Hn to the tops of the p l a n t s , which can lead to Hn t o x i c i t y (Foy 1983). However, an i n v e r s e r e l a t i o n s h i p was found between Zn a p p l i c a t i o n and Hn i n soybean (White er al. 1979), maize (Singh and Steenberg 1974), and bush beans (Ruano er al. 1987). 161 Since there appeared to be no antagonism between f o l i a r Zn c o n c e n t r a t i o n s and Mn a p p l i c a t i o n s or f o l i a r Mn c o n c e n t r a t i o n s and Zn a p p l i c a t i o n s , the evidence from t h i s study i n d i c a t e s that i t i s u n l i k e l y the lower f o l i a r Zn l e v e l s i n these hemlock stands are due to Mn antagonism. The evidence from the l i t e r a t u r e suggests an antagonism between Zn and Mn (Reddy et al. 1978; Nair et al. 1977; Malav o l t a er al. 1956; Hawf er al. 1967). However, i n agreement with the r e s u l t s i n t h i s study, Kohno er al. (1984) found i n bean p l a n t s and Shuman and Anderson (1976) found i n soybeans that the f o l i a r l e v e l s of Zn i n c r e a s e d with i n c r e a s i n g Mn c o n c e n t r a t i o n s i n the s o l u t i o n . I t i s i n t e r e s t i n g that s o i l a p p l i c a t i o n s of Zn d i d not in c r e a s e f o l i a r Zn a f t e r the f i r s t year but i n some cases s o i l a p p l i c a t i o n s of Mn d i d . Since there was a p o s i t i v e growth response with Mn i n some cases t h i s may have c r e a t e d a si n k f o r Zn. T h i s may suggest an i n c r e a s e d Zn requirement with Mn. With s i m i l a r s o i l a p p l i c a t i o n l e v e l s of Zn and Mn (200 kg h a - 1 ) a g r e a t e r percentage of Mn was found i n the f o l i a g e compared to the Zn treatment. This suggests d i f f e r e n t s o i l chemical r e a c t i o n s or p l a n t u p t a k e / t r a n s l o c a t i o n mechanisms f o r the two elements. D i f f e r e n t r e t e n t i o n and/or r e l e a s e mechanisms of the s o i l f o r Zn and Mn may be r u l e d out because hemlock had d i f f e r e n t f o l i a r l e v e l s of Zn and Mn i n comparison to other tree s p e c i e s i n the same stands. T h i s would i n d i c a t e that the d i f f e r e n c e s between f o l i a r l e v e l s of the two elements i s due to 162 p l a n t f a c t o r s such as d i f f e r e n c e s i n n u t r i e n t uptake or i n t r a n s l o c a t i o n . Nair and Prabhat (1977), found that with i d e n t i c a l a p p l i c a t i o n r a t e s , r e l a t i v e l y more Zn was root absorbed than Mn, but of the amount root absorbed, r e l a t i v e l y more Mn was t r a n s l o c a t e d to the shoots. T h i s would suggest d i f f e r e n t mechanisms of t r a n s l o c a t i o n f o r Zn and Mn. An i n t e r a c t i o n was found between f o l i a r Zn and f o l i a r N c o n c e n t r a t i o n s . An i n t e r a c t i o n between Zn and N i n t r e e s has been found i n s e v e r a l other s t u d i e s . R e s u l t s of Zasoski and Porada (1986) i n d i c a t e a s t r o n g r e l a t i o n s h i p between N and Zn i n hemlock growing on s l a s h b u r n t s i t e s . F o l i a r l e v e l s were <1.1 cg g _ l N and <12 pg g - i Zn, which according to a DRIS a n a l y s i s were s t r o n g l y growth l i m i t i n g . High a p p l i c a t i o n s of N d i d not s t i m u l a t e growth nor i n c r e a s e f o l i a r N l e v e l s , which suggested that Zn was inadequate f o r N. McGrath and Robson (1984) i n v e s t i g a t e d the e f f e c t of N and P supply on the response of P. radiata to Zn. They found that a response to Zn depended on the requirements f o r N and P being p r o v i d e d . Evidence from these e m p i r i c a l s t u d i e s i m p l i e s an i n t e r a c t i o n between Zn and N. There i s evidence i n the l i t e r a t u r e to support a f u n c t i o n a l r e l a t i o n s h i p between Zn and N. F i r s t l y , t h i s i n t e r a c t i o n may occur because of the e f f e c t of one element on the t r a n s p o r t of the other element i n the s o i l or the p l a n t . For example t r a n s i t i o n metals such as Zn have high a f f i n i t i e s f o r -M-ligands (Robson and Pitman 1983). 163 The second way i n which t h i s i n t e r a c t i o n may occur i s i n p r o t e i n s y n t h e s i s . There are three mechanisms by which Zn a f f e c t s p r o t e i n s y n t h e s i s . Nitrogen i s known to be an e s s e n t i a l component of p r o t e i n s . Zinc d e f i c i e n c y causes a r e d u c t i o n i n p r o t e i n and ribosomal RNA contents ( K i t a g i s h i and Obata 1986. 1987; Obata and Umebayashi 1988; Prask and Plocke 1971; Wacker 1962; Sharam er al. 1981). C o n c u r r e n t l y there i s an accumulation of amino a c i d s and amides ( K i t a g i s h i and Obata 1986; Wacker 1962; Possingham 1956). The f i r s t mechanism i n v o l v e s the t r a n s c r i p t i o n of DNA by RNA. Zinc forms chemical bonds with the amino a c i d s c y s t i n e and h i s t i d i n e i n such a way that the c h a i n of amino ac i d s becomes f o l d e d around the z i n c to form a loop or ' z i n c f i n g e r * (Figure 27). P r o t e i n s (RNA polymerase) that r e g u l a t e the t r a n s c r i p t i o n of DNA to RNA do so through t h e i r z i n c f i n g e r s . If the z i n c i s absent, the p r o t e i n cannot bind to DNA and r e g u l a t e i t s t r a n s c r i p t i o n (Parraga er al. 1988). T h e r e f o r e , z i n c i s e s s e n t i a l i n terms of i t s s t r u c t u r a l r o l e . The second way i s through the occurrence of Zn i n the t o t a l RNA f r a c t i o n , and i t s e f f e c t on the ribosomes; Zn may be r e q u i r e d to maintain the s t r u c t u r a l i n t e r g r i t y of the ribosomes (Prask and Plocke 1971). 164 F i g u r e 2 7 . R i b b o n m o d e l o f a s i n g l e z i n c f i n g e r d o m a i n ( A D R l a ) i n c o r p o r a t i n g t e t r a h e d r a l c o o r d i n a t i o n o f z i n c by c y s t i n e ( C ) and h i s t i d i n e (H) ( f r o m P a r r a g a et al. ( 1 9 8 8 ) ) . 165 The t h i r d mechanism i n v o l v e s the r e g u l a t i o n of RNA degradation by Zn. Higher r a t e s of RNAase a c t i v i t y are observed with Zn d e f i c i e n c y (Marschner 1986). This demonstates the importance of Zn f o r p r o t e i n s y n t h e s i s . F. F o l i a r A p p l i c a t i o n of Zn i n F o r e s t r y F o l i a r - a p p l i e d sprays are the most e f f i c i e n t means of s u p p l y i n g hemlock t r e e s with Zn. T h i s method would appear to be of i m p r a c t i c a l use i n l a r g e r f e r t i l i z a t i o n experiments or i n o p e r a t i o n a l f e r t i l i z a t i o n of stands. However, p e s t i c i d e s i n s o l u t i o n form are r o u t i n e l y a p p l i e d i n o p e r a t i o n a l f o r e s t r y with fixed-winged a i r c r a f t and h e l i c o p t e r s . T h i s same technology could be a p p l i e d to the f o l i a r a p p l i c a t i o n of n u t r i e n t s . 166 CHAPTER 6 . CONCLUSIONS T h i s s t u d y was u n d e r t a k e n i n o r d e r t o i n v e s t i g a t e t h e t h e s i g n i f i c a n c e o f t h e c h a r a c t e r i s t i c p a t t e r n o f f o l i a r Zn and Mn c o n c e n t r a t i o n s o f h e m l o c k i n i t s n u t r i t i o n . Two a p p r o a c h e s t o t h e s t u d y w e r e u s e d : c o m p a r a t i v e n u t r i t i o n and t h e s c r e e n i n g t r i a l - g r o w t h r e s p o n s e t e c h n i q u e . I n a c o m p a r i s o n o f t o t a l f o l i a r c o n c e n t r a t i o n s , h e m l o c k had l o w e r Zn c o m p a r e d t o D o u g l a s - f i r , a m a b i l i s f i r and w h i t e p i n e . I n c o n t r a s t , h e m l o c k had h i g h e r Mn c o m p a r e d t o D o u g l a s - f i r , a m a b i l i s f i r , w h i t e p i n e , r e d c e d a r and y e l l o w c e d a r . A n a l y s i s o f c e l l u l a r f r a c t i o n s o f f o l i a g e p r o d u c e d two r e s u l t s . F i r s t , z i n c and manganese a c c u m u l a t e d i n two d i f f e r e n t f r a c t i o n s i r r e s p e c t i v e o f t h e l e v e l o f t h e t r e a t m e n t o r t h e s p e c i e s . A c c u m u l a t i o n i n c e r t a i n f r a c t i o n s may i n d i c a t e a p h y s i o l o g i c a l n e e d i n t h a t f r a c t i o n o r a t o l e r a n c e m e c h a n i s m . S e c o n d , c o m p a r i n g h e m l o c k t o D o u g l a s - f i r t o t a l Zn l e v e l s t e n d t o be c o n s i s t e n t w i t h l e v e l s i n d i f f e r e n t f r a c t i o n s , i n d i c a t i n g t o t a l l e v e l s may be an a d e q u a t e i n d i c a t i o n o f p h y s i o l o g i c a l l e v e l s . C o m p a r i n g h e m l o c k t o D o u g l a s - f i r , t o t a l Mn l e v e l s a r e c o n s i s t e n t w i t h Mn l e v e l s i n d i f f e r e n t f r a c t i o n s , i n d i c a t i n g h e m l o c k may h a v e a g r e a t e r p h y s i o l o g i c a l Mn r e q u i r e m e n t o r a Mn t o l e r a n c e m e c h a n i s m . 167 There were both a n u t r i e n t c o n c e n t r a t i o n response and a growth response to z i n c a p p l i c a t i o n s . The timing of response was dependent upon the method of a p p l i c a t i o n . Response to f o l i a r a p p l i c a t i o n s occurred i n the f i r s t year f o l l o w i n g treatment, and response to s o i l a p p l i c a t i o n s occurred i n the second year f o l l o w i n g treatment. Growth response as measured by shoot increment r a t i o was obtained p r i m a r i l y i n the second year a f t e r treatment to f o l i a r a p p l i c a t i o n s of z i n c . Height increment r a t i o i n c r e a s e d i n response to f o l i a r z i n c a p p l i c a t i o n s i n the second year. The f a c t t h at there were cases of p o s i t i v e growth responses to Zn f e r t i l i z a t i o n i s evidence that the r e l a t i v e l y lower f o l i a r Zn c o n c e n t r a t i o n s of hemlock compared to some other c o n i f e r s do i n d i c a t e some d e f i c i e n c y of Zn i n the stands used f o r t h i s study. A d d i t i o n a l evidence of a Zn d e f i c i e n c y are; the higher f o l i a r Zn l e v e l s a s s o c i a t e d with optimum height increment compared to c o n t r o l f o l i a r Zn l e v e l s , r e t r a n s l o c a t i o n of Zn i n the second year f o l l o w i n g the high f o l i a r Zn treatment and the high ranking of Zn i n the vect o r graphs from the "complete-Zn-Mn" treatment. In some cases, r a t h e r than a growth response to Zn, lu x u r y consumption of Zn oc c u r r e d . For p l a n t s having low n u t r i e n t requirements, l u x u r y consumption i s a p h y s i o - e c o l o g i c a l mechanism which s u p p l i e s the p l a n t during p e r i o d s of n u t r i e n t i n s u f f i c i e n c y between the p u l s e s of lu x u r y a v a i l a b i l i t y . A s i m i l a r s i t u a t i o n 168 may operate f o r hemlock, s i n c e t h i s s p e c i e s has low n u t r i e n t requirements. There were both p o s i t i v e n u t r i e n t and growth responses to s o i l Hn treatments. P o s i t i v e n u t r i e n t c o n c e n t r a t i o n and growth responses occurred i n the f i r s t year. F o l i a r Hn was s t i l l e l e v a t e d i n the second year i n the one-year-old and two-year-old f o l i a g e . The e l e v a t e d f o l i a r Hn i n the second year must have been from i n c r e a s e d uptake from the s o i l or t r a n s l o c a t i o n from the r o o t s , s i n c e there was no r e t r a n s l o c a t i o n from the two-year- o l d f o l i a g e . Hemlock has f o l i a r Hn l e v e l s which are considered t o x i c i n some p l a n t s , and the f a c t t h at hemlock continued to take up s o i l - a p p l i e d Hn with p o s i t i v e growth responses i n d i c a t e that hemlock i s Hn - t o l e r a n t and a l s o has i n c r e a s e d requirements f o r Hn. The f o l i a r Hn l e v e l s may not adequately r e f l e c t the p h y s i o l o g i c a l requirements f o r Hn. There are both a p h y s i o l o g i c a l component and an e c o l o g i c a l component of a p l a n t ' s n u t r i t i o n . For Hn, there may be two p h y s i o l o g i c a l pathways i n hemlock. One pathway may be a c t i v e i n complexing the metal, o p e r a t i n g as a t o l e r a n c e mechanism, g i v i n g hemlock a competitive advantage i n a c i d s o i l . The second pathway may channel the metal i n t o the growth metabolism of the p l a n t . How the a l l o c a t i o n of manganese i s r e g u l a t e d between the two competing paths i s not known. In general t h i s s i t u a t i o n may apply to m e t a l - t o l e r a n t p l a n t s where a metal on the one hand i s being d e t o x i f i e d by the 169 t o l e r a n c e mechanism, but on the other hand i s r e q u i r e d i n growth metabolism. There must be a mechanism which r e g u l a t e s the a l l o c a t i o n of the metal between the two pathways. This t o p i c which i n v o l v e s both e c o l o g i c a l and p h y s i o l o g i c a l p l a n t n u t r i t i o n r e q u i r e s g r e a t e r i n v e s t i g a t i o n . Determining the n u t r i e n t s t a t u s of a m i c r o n u t r i e n t i n a p l a n t which i s an accumulator of the m i c r o n u t r i e n t may be p r o b l e m a t i c . I t i s necessary to determine the p h y s i o l o g i c a l l y a c t i v e f r a c t i o n of the n u t r i e n t . T h i s may be done using e x t r a c t s of the n u t r i e n t whose l e v e l i s c o r r e l a t e d to the r a t e of a p h y s i o l o g i c a l a c t i v i t y , or s e p a r a t i o n and a n a l y s i s of c e l l u l a r f r a c t i o n s which are i n v o l v e d i n a p h y s i o l o g i c a l p r o c e s s . F o l i a r a p p l i c a t i o n of Zn and s o i l a p p l i c a t i o n of Mn appeared to be the most e f f i c i e n t means of s u p p l y i n g the p l a n t with these n u t r i e n t s . Increased f o l i a r l e v e l s of Zn d i d occur with s o i l Zn treatments but t h i s was delayed u n t i l the second year a f t e r f e r t i l i z a t i o n . It i s hypothesized that t h i s i s due to i n h i b i t e d uptake and/or t r a n s l o c a t i o n of Zn. Low f o l i a r Zn c o n c e n t r a t i o n s i n hemlock are not due to low i n h e r e n t z i n c f e r t i l i t y of the s o i l , s i n c e s p e c i e s which c o e x i s t with hemlock i n the same stands tend to have higher f o l i a r z i n c . A growth response ( f o l i a r mass per shoot and shoot increment r a t i o ) was obtained with the "complete-Zn-Mn" treatment. Vector a n a l y s i s which ranked the r e l a t i v e responses 170 r e v e a l e d the e x i s t e n c e of n u t r i e n t d e f i c i e n c i e s other than Zn and Mn. Vector a n a l y s i s a l s o r e v e a l e d evidence of a strong Zn d e f i c i e n c y . F o l i a r Zn was s y n e r g i s t i c to the "complete-Zn-Mn" treatment i n both the f i r s t and second years a f t e r treatment. T h e r e f o r e , i t was d i f f i c u l t to say whether the growth response was due to the a p p l i e d n u t r i e n t s , the Zn, or an improved balance of a l l n u t r i e n t s . Conceivably, f e r t i l i z a t i o n could lead to a Zn d e f i c i e n c y i f the n a t i v e supply i n the s o i l cannot meet the i n c r e a s e d demand by the p l a n t . Such a s i t u a t i o n i s termed an induced d e f i c i e n c y . Induced Zn d e f i c i e n c y or other n u t r i e n t d e f i c i e n c i e s may be a d d i t i o n a l reasons why growth response i n hemlock to n i t r o g e n f e r t i l i z a t i o n has been i n c o n s i s t e n t . Ingestad's n u t r i e n t r a t i o s were c a l c u l a t e d f o r the f o l i a r l e v e l s from the c o n t r o l and the "complete-Zn-Mn" treatments. Comparing these r a t i o s to the optimum reve a l e d that most of the n u t r i e n t s were i n balance except f o r i r o n and manganese. E x i s t i n g d i a g n o s t i c norms for Zn appear to adequately d e s c r i b e the Zn n u t r i t i o n of hemlock. Response to f e r t i l i z a t i o n occurred with c o n t r o l f o l i a r Zn c o n c e n t r a t i o n s f o r hemlock being below the c r i t i c a l l e v e l of 15 pg g ~ x . D i a g n o s t i c norms f o r Mn need to be r e v i s e d . Response occurred even though c o n t r o l f o l i a r Mn c o n c e n t r a t i o n s f o r hemlock were well above the c r i t i c a l l e v e l of 25 pg g - 1 . T h e r e f o r e , t o t a l f o l i a r Mn may not be i n d i c a t i v e of the p h y s i o l o g i c a l Mn s t a t u s of hemlock. 171 The e m p i r i c a l r e l a t i o n s h i p found between f o l i a r Zn and N suggests an i n t e r a c t i o n between the two elements. The response of the pl a n t to the a d d i t i o n of one of these elements i s dependent upon the adequate supply of the other element. This i n t e r a c t i o n r e q u i r e s f u r t h e r i n v e s t i g a t i o n to determine i f v a r i a b i l i t y i n response to N f e r t i l i z a t i o n may be a f f e c t e d by v a r i a b i l i t y i n the Zn s t a t u s of the p l a n t . F o l i a r Zn was r a t h e r than being a n t a g o n i s t i c was i n some cases s y n e r g i s t i c to s o i l a p p l i c a t i o n s of Mn . Therefore, there was no evidence i n t h i s study to suggest that low f o l i a r l e v e l s of Zn i n hemlock are due to a Mn antagonism. The onl y i n t e r a c t i o n obtained with the "complete-Zn -Mn" treatment was a synergism with f o l i a r Zn. There are s e v e r a l aspects of t h i s research which r e q u i r e f u r t h e r . i n v e s t i g a t i o n . For the i n f o r m a t i o n from these sc r e e n i n g t r i a l s to be a p p l i e d to the management of hemlock, s e v e r a l f u r t h e r steps need to be taken. The next step f o l l o w i n g the a n a l y s i s of s c r e e n i n g t r i a l s would be s t u d i e s of c o r r e l a t i o n between s i t e f a c t o r s and responsiveness. T h i s i n f o r m a t i o n would then be used f o r c l a s s i f y i n g o p e r a t i o n a l stands i n t o response c a t e g o r i e s . Another aspect which needs i n v e s t i g a t i n g i s the i n t e r a c t i o n between n i t r o g e n n u t r i t i o n and z i n c . Since there i s evidence of d e f i c i e n c i e s of Zn, Mn and other m i c r o n u t r i e n t s optimum n u t r i t i o n experiments need to i n c l u d e these. T h i s may be done by s u p p l y i n g optimum dosages of macronutrients wit'h 172 d i f f e r e n t dosages of i n d i v i d u a l m i c r o n u t r i e n t s or d i f f e r e n t balances of m i c r o n u t r i e n t s . Manganese n u t r i t i o n of hemlock may be s i m i l a r to that of other metal-accumulating p l a n t s i n that there i s a high requirement f o r the metal. T h i s would i n v o l v e i n v e s t i g a t i o n of the r e g u l a t i o n of Mn between the two p h y s i o l o g i c a l pathways of m e t a l - t o l e r a n c e and growth. Luxury consumption may be a c h a r a c t e r i s t i c of p l a n t s having low n u t r i e n t requirements. 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D i s c r i m i n a n t a n a l y s i s i n tr e e n u t r i t i o n r e s e a r c h . For. S c i . 17:425-427. 187 White, H.C., R.L. Chaney, and A.M. Decker. 1979. D i f f e r e n t i a l c u l t i v a r t o l e r a n c e i n soybean to p h y t o t o x i c l e v e l s of s o i l Zn. I I . Range of Zn a d d i t i o n s and the uptake and t r a n s l o c a t i o n of Zn, Mn, Fe, and P. Agron. J . 71:126-131. Woolhouse, H.W. 1983. T o x i c i t y and t o l e r a n c e i n response of p l a n t s to metals. In E n c y c l o p e d i a of p l a n t p h y s i o l o g y . Edited by O.L. Lange, V o l . 12C. S p r i n g e r - V e r l a g , New York. pp. 246- 300. Zar, J.H. 1984. B i o s t a t i s t i c a l a n a l y s i s . P r e n t i c e - H a l l , Inc., Englewood C l i f f s , N.J. 620 pp. Z a s o s k i , R.J., and S.P. G e s s e l . 1982. Response of western hemlock s e e d l i n g s to N, P, and S f e r t i l i z a t i o n i n a c o a s t a l Washington s o i l . Agron. A b s t r . 273. Z a s o s k i , R.J., and H.J. Porada. 1986. Tissue n u t r i e n t s t a t u s and DRIS as i n d i c a t o r s of outplanted D o u g l a s - f i r and western hemlock growth. Agron. A b s t r . 270. Z a s o s k i , R.J., S.G. A r c h i e , W.C. Swain, and J.D. S t e d n i c k . 1977. The impact of sewage sludge on D o u g l a s - f i r stands near Port Gamble. Wash. C o l l . For. Res. Univ. Wash. Z a s o s k i , R.J., H.J. Porada, P.J. Ryan, J . G r e e n l e a f - J e n k i n s , and S.P. G e s s e l . 1990. Observations of copper, z i n c , i r o n and manganese s t a t u s i n western Washington f o r e s t s . For. E c o l . Manage. 37:7-25. Z a s o s k i , R.J., R.L. Edmonds, C S . Bledsoe, C.L. Henery, D.J. Vogt, K.A. Vogt, and D.W. C o l e . 1984. M u n i c i p a l sewage sludge use i n f o r e s t s of the P a c i f i c Northwest, U.S.A.: Environmental Concerns. Waste. Manage. Res., 2:227-246. 188 APPENDIX A. SITE AND SOIL DESCRIPTION APPENDIX A l . SITE I NTS Sheet: Port Coquitlam 92/G7 L o c a t i o n : UBC Research For e s t Road: F70 Long: 122* 34' 10" L a t : 49* 17' 40" E l e v a t i o n : 360 m Slope: Very gentle s l o p e s 2-5% Aspect: North-west Landform: T i l l over bedrock Parent M a t e r i a l : T i l l Drainage C l a s s : Well Perviousness: Moderate Depth of P i t : 52 cm Rooting: 52 cm Depth to Water Table: Never Present Compact T i l l : 45 cm Bedrock: 52 cm S o i l C l a s s i f i c a t i o n : O r t h i c Humo-Ferric Podzol Humus Form: Humimor BGCZ: Windward Submontane Maritime C o a s t a l Western Hemlock Wetter CWHbl Major V e g e t a t i o n : Tsuga beterophylla, Abies amabilis, Pseudotsuga menziesii (planted) Lower V e g e t a t i o n : Vaccinium parvifolium, Hylocomium splendens, Vaccinium alaskaense, Blechnum spi cant. 189 S o i l P r o f i l e LFH 3-0 Black (10 YR 3/1, m o i s t ) ; f r e s h and p a r t i a l l y decomposed o r g a n i c matter; c l e a r and i r r e g u a l r boundary, 3-5 cm t h i c k . Bf 0-42 Dark y e l l o w i s h brown (10 YR 4/4, m o i s t ) ; loamy sand; s i n g l e g r a i n e d ; f i n e sub-angular blocky; s l i g h t l y s t i c k y , s l i g h t l y p l a s t i c , very f r i a b l e ; few f i n e and coarse r o o t s ; about 40% coarse fragments; abrupt, i r r e g u l a r boundary; C 42-52 Brown (10 YR 5/3, m o i s t ) ; loamy sand; s i n g l e d g r a i n e d ; f i n e - c o a r s e sub-angular blocky; s l i g h t l y s t i c k y , s l i g h t l y p l a s t i c , very f i r m ; very few medium and coarse r o o t s ; about 40% coarse fragment content; abrupt, i r r e g u l a r boundary; 190 APPENDIX A2. SITE 2 NTS Sheet L o c a t i o n : Road: Long: Lat : E l e v a t i o n Slope: Aspect: None Landform: T i l l Parent M a t e r i a l Drainage C l a s s : Perviousness: Depth of P i t : Rooting: Depth to Water Port Coquitlam 92/G7 UBC Research Forest E12 122° 34' 10" 49° 18' 50" 440 m Very gentle s l o p e s 2-5% over bedrock : T i l l I m p e r f e c t l y Drained Slowly pervious 43 cm 43 cm Table: Never Present Compact T i l l : None Bedrock: 43 cm S o i l C l a s s i f i c a t i o n : Rego G l e y s o l Humus Form: Humimor BGCZ: Windward Submontane Maritime C o a s t a l Western Hemlock Wetter CWHbl Major V e g e t a t i o n : Tsuga heterophylla, monticola Lower V e g e t a t i o n : Vaccinium parvifolium, NU yl. i if i ti litil.phu n lorn u Thuja plicata, Pinus Gaultheria Ki. nit ft if r,if J. n shallon, oregana 191 S o i l P r o f i l e LFH 15-0 Black (10 YR 3/1, m o i s t ) ; f r e s h and p a r t i a l l y decomposed or g a n i c matter; abundant very f i n e and f i n e , and few medium r o o t s ; abrupy, wavy boundary; 15-20 cm t h i c k . Cg 0-18 Gray (10 YR 5/1, mo i s t ) ; loamy sand; s i n l e g r a ined; moderatley, f i n e , subangular blocky; s l i g h t l y s t i c k y , s l i g h t l y p l a s t i c , very f r i a b l e , s o f t ; p l e n t i f u l very f i n e and f i n e r o o t s ; about 30% coarse fragments; abrupt and wavy boundary; 7-20 cm t h i c k . APPENDIX A3. SITE 3 NTS Sheet: C h i l l i w a c k 92/H4 L o c a t i o n : Chipmunk Creek Road: Chipmunk.creek Road Long: 121° 41' 50" L a t : 49° 8' 10" E l e v a t i o n : 880-960 m Slope: Very s t r o n g s l o p e s 31-45% Aspect: South-east Landform: T i l l over bedrock Parent M a t e r i a l : T i l l Drainage C l a s s : R a p i d l y Drained Perviousness: R a p i d l y pervious Depth of P i t : 1.5 m Rooting: 60 cm Depth to Water Table: Never Present Compact T i l l : 60 cm Bedrock: None S o i l C l a s s i f i c a t i o n : O r t h i c Humo-Ferric P o d 2 o l Humus Form: Humimor BGCZ: Windward Montane Maritime C o a s t a l Western Hemlock Wetter CWHb2 Major V e g e t a t i o n : Tsuga heteropbylla, Abies amabilis, Pseudotsuga menziesii (planted) Lower V e g e t a t i o n : Rhytidiopsis robusta, Vaccinium alaskaense, Blecbnum spicant, Moneses uniflora 193 Appendix A3 (continued) S o i l P r o f i l e L 2-0 Black (10 YR 3/1, m o i s t ) ; f r e s h and p a r t i a l l y decomposed or g a n i c m a t e r i a l ; abundant f i n e and very f i n e r o o t s ; abrupt, broken boundary. Ae 0-5 Gray (10 YR 6/1, m o i s t ) ; loamy sand; s i n g l e g r a i n e d ; weak, f i n e - c o a r s e subangular blocky; n o n s t i c k y , n o n p l a s t i c , l o o s e , s o f t ; abundant very f i n e - f i n e r o o t s ; about 5% coarse fragments; abrupt, broken boundary; 0-5 cm t h i c k . Bf 5-60 Y e l l o w i s h Brown (10 YR 5/6, m o i s t ) ; loamy sand; s i n g l e g r a i n e d ; moderately, f i n e - c o a r s e subangular blocky; s l i g h t l y s t i c k y , s l i g h t l y p l a s t i c , very f r i a b l e , s o f t ; abundant f i n e - medium, and very few coarse r o o t s ; about 30% coarse fragment content; gradual wavy boundary; 45-60 cm t h i c k . Cg 60-*- Gray (10 YR 6/1, m o i s t ) ; coarse sand; s i n g l e g r a i n e d ; stong, f i n e - c o a r s e subangular blocky; s l i g h t l y s t i c k y , s l i g h t l y p l a s t i c , very f r i a b l e , s o f t ; about 50% coarse fragment content. 194 APPENDIX A4. SITE 4 NTS Sheet: Port Coquitlam 92/G7 L o c a t i o n : UBC Research Forest Road: E13 Long: 122° 34' 20" La t : 49° 18' 50" E l e v a t i o n : 520 m Slope: Nearly l e v e l 0.5-2% Aspect: North Landform: T i l l over bedrock Parent M a t e r i a l : T i l l Drainage C l a s s : Well Drained P e r v i o u s n e s s : Moderately pervious Depth of P i t : 90 cm Rooting: 70 cm Depth to Water Table: Never Present Compact T i l l : 80 cm Bedrock: None S o i l C l a s s i f i c a t i o n : Duric Humo-Ferric Podzol Humus Form: Humimor BGCZ: Windward Submontane Maritime C o a s t a l Western Hemlock Wetter CWHbl Major V e g e t a t i o n : Tsuga heterophylla, Abies amabilis, Pseudotsuga menziesii (planted) Lower V e g e t a t i o n : Vaccinium parvifolium, Gaultheria shallon, Rhytidiadelphus loreus, Kindbergia oregana 195 S o i l P r o f i l e LFH 20-0 Black (10 YR 3/1, m o i s t ) ; f r e a s h , p a r t i a l l y and well decomposed o r g a n i c m a t e r i a l ; abundant f i n e and very f i n e , and few coarse r o o t s ; gradual smooth boundary; 5-20 cm t h i c k . Ae 0-10 Gray (10 YR 6/1, mo i s t ) ; loamy sand; s i n g l e g r ained; moderately, medium, subangular blocky; s l i g h t l y s t i c k y , s l i g h t l y p l a s t i c , very f r i a b l e , s o f t ; abundant f i n e and medium r o o t s ; about 30% coarse fragments; c l e a r and i r r e g u l a r boundary; 0-10 cm t h i c k . Bf 10-46 Dark y e l l o w i s h brown (10 YR 4/4, m o i s t ) ; loamy sand; s i n g l e g r a i n e d ; moderately, medium, subangular blocky; s l i g h t l y s t i c k y , s l i g h t l y p l a s t i c , very f r i a b l e , s o f t ; few f i n e and medium r o o t s ; about 30% coarse fragment content; c l e a r and wavy boundary; 36 cm t h i c k BCc 46-72 Y e l l o w i s h red (5 YR 5/8, m o i s t ) ; loamy sand; s i n g l e g r a i n e d ; moderately, medium, subangular blocky; s l i g h t l y s t i c k y , s l i g h t l y p l a s t i c , very f r i a b l e , s o f t ; about 30% coarse fragment content; c l e a r and wavy boundary; 10-26 cm t h i c k ; C 72+ Y e l l o w i s h brown (10 YR 5/6, m o i s t ) ; coase sand; s i n g l e g r a i n e d ; moderately, medium, subangular blocky to p l a t y ; s l i g h t l y s t i c k y , s l i g h t l y p l a s t i c , very f r i a b l e , s o f t ; about 50% coarse fragment content. APPENDIX A5. SITE 5 NTS Sheet: Stave Lake 93/G6 L o c a t i o n : M i s s i o n Tree Farm Road: Rock Creek Road Long: 122° 34' 20" La t : 49° 18' 50" E l e v a t i o n : 1,100-1,200 m Slope: - Very Strong Slopes 31-45% Aspect: East Landform: T i l l over bedrock Parent M a t e r i a l : T i l l Drainage C l a s s : Well Drained Perviousness: Moderately pervious Depth of P i t : 80 cm Rooting: 40 cm Depth to Water Table: Never Present Compact T i l l : 80 cm Bedrock: None S o i l C l a s s i f i c a t i o n : O r t h i c Ferro-Humic Podzol Humus Form: Humimor BGCZ: Windward Montane Maritime C o a s t a l Western Hemlock Wetter CWHb2 Major V e g e t a t i o n : Tsuga heterophylla, Abies amabilis, Chamaecyparis nootkatensis, Pseudotsuga menziesii (planted) Lower V e g e t a t i o n : Vaccinium alaskaense, Dryopteris expansa, Blecbnum spicant, Sphagnum gicgensohnd-i, Tiarclla uni f o l i a t a 197 Appendix AS (continud) S o i l P r o f i l e LFH 25-0 Black (10 YR 3/1, m o i s t ) ; f r e s h , p a r t i a l l y , and w e l l decompose or g a n i c m a t e r i a l ; abundant very f i n e and f i n e , few coarse, and p l e n t i f u l medium; abrupt and smooth boundary; 20-25 cm t h i c k . Bhf 0-18 Black (10 YR 3/1, m o i s t ) ; loamy sand; s i n g l e p a r t i c l e s ; s t r o n g , f i n e - v e r y coarse subangular blocky; s t i c k y , p l a s t i c , very f i r m , s o f t ; p l e n t i f u l f i n e , and medium r o o t s ; about 20% coarse fragment content; abrupt, smooth boundary; 18-20 cm t h i c k . Bf 18-35 Dark y e l l o w i s h brown (10 YR 4/4, m o i s t ) ; loamy sand; s i n g l e p a r t i c l e s ; s t r o n g , f i n e - v e r y coarse subangular blocky; s t i c k y , p l a s t i c , very f i r m , s o f t ; very few f i n e , and medium r o o t s ; about 402 coarse fragment content; abrupt, smooth boundary; 17-20 cm t h i c k . C 35+ L i g h t gray (7.5 YR 7/0, m o i s t ) ; sand; s i n g l e g r a i n e d ; red (2.5 YR 4/8, moist) mottles; n o n s t i c k y , n o n p l a s t i c , very f r i a b l e , s o f t ; about 50% coarse fragment content. 198 APPENDIX B l . MODIFIED PARKINSON AND ALLEN DIGESTION FOR PLANT TISSUE ANALYSIS 1) Weigh 1 g (to the nearest mg) subsample of oven-dried (70°C f o r 3 hours), ground f o l i a g e and place i n 100 ml d i g e s t i o n tube. 45 tubes i n each set can be prepared, with a r e f e r e n c e sample and a blank i n each s e t . 2) Add 5 ml of cone. HaSO* (reagent grade) to each sample, and mix on a mechanical v i b r a t o r immediately. 3) Dispense 1 ml of LiaSO* - HaOa mixture (prepared by mixing 7.0 g L 1 2 S 0 A , 0.21 g selenium powder i n 175 ml 30% HaOa) i n t o each tube. Wait u n t i l r e a c t i o n (foaming and s p a t t e r i n g ) ceases before c o n t i n u i n g . 4) Repeat step 3. 5) Heat the rack of tubes on the d i g e s t i o n block at 360°C. Use d i s c o n t i n u o u s heating to overcome i n i t i a l foaming; that i s , 20-40 seconds on block, c o o l f o r about 2 minutes, 40-50 seconds of heating and c o o l , 1-2 minutes on block and c o o l f o r 5-10 minutes. 6) Add another 1 ml LiaSO* - HaOa mixture to each tube. Wait t i l l r e a c t i o n ceases. 7) Repeat step 6. 8) Digest on block f o r 1 1/2 hours at 360°C. 9) A f t e r 1 1/2 hours, remove rack from block. Add 0.5 ml HaOa to each tube, r e t u r n rack to block and d i g e s t f o r another 30 minutes. 10) Repeat step 9. T o t a l d i g e s t i o n time i s 2 1/2 hours. 11) Remove rack from block and allow d i g e s t s to c o o l (approximately 1 hour). Samples should be pale yellow to milky white i n c o l o u r . 12) Add about 80 ml of d e m i n e r a l i z e d water. Allow to c o o l to room temperature before making to a f i n a l volume (100 ml) with d e m i n e r a l i z e d water. 13) Cover tubes with an i n e r t stopper, i n v e r t 3-4 times to mix, and pour contents i n t o a l a b e l l e d 125 ml p l a s t i c b o t t l e . 199 APPENDIX B2. NITRIC ACID DIGESTION FOR ANALYSIS OF COPPER AND IRON IN FOLIAGE 1) Weigh 0.7 g (to the nearest mg) subsample of oven-dried (70°C f o r 3 hours), ground f o l i a g e and p l a c e sample i n t o d i g e s t i o n tube. A set of 45 tubes can be prepared f o r one run, each set having a r e f e r e n c e sample and a blank. 2) Add 5 ml cone. HNOa to sample, and mix on mechanical v i b r a t o r . 3) Cover the tubes with g l a s s marbles and heat on d i g e s t i o n block at 40°C f o r 1 hour. 4) Increase heat up to 140°C and continue heating f o r 2 hours, c o u t i n g from time the block reaches 140*0. 5) Remove tubes from block and allow to c o o l . 6) Add about 7 ml d e m i n e r a l i z e d water to each tube and mix by s w i r l i n g . Allow to c o o l . Pour sample i n t o a 25 ml measuring c y l i n d e r . Rinse d i g e s t i o n tube with d e m i n e r a l i z e d water and add r i n s i n g s to the c y l i n d e r . Make volume up to 25 ml with d e m i n e r a l i z e d water. Cover c y l i n d e r with an i n e r t rubber stopper and mix content by i n v e r t i n g 3-4 times. T r a n s f e r contents to a 60 ml p l a s t i c b o t t l e . 7) Analyze the s o l u t i o n s f o r i r o n and copper by atomic a b s o r p t i o n spectrophotometry. . 200 APPENDIX B3. PROCEDURE FOR THE DETERMINATION OF SULPHATE-SULPHUR IN FOLIAGE E x t r a c t i o n from f o l i a g e 1) Weigh one gram of oven-dried f o l i a g e i n t o an erlenmeyer f l a s k . Add 20 ml of 1 N HCL and record weight. 2) Heat on a hot p l a t e to b o i l i n g , then continue b o i l i n g f o r 10 minutes. 3) Add water to b r i n g back to the o r i g i n a l weight. 4) F i l t e r through #42 f i l t e r paper. D i g e s t i o n 1) Use from 0.5 to 2 ml of sample. 2) Place 5 ml of 1 N NaOH i n t o a 20 ml t e s t tube and attach so the d e l i v e r y tube reaches almost to the bottom of the t e s t - t u b e . 3) Add 4 ml of reducing agent (mix 300 ml of h y d r i o d i c a c i d (57% and 1% p r e s e r v a t i v e ) with 75 ml of 50% hypophosphorous a c i d and 150 ml of 90% formic a c i d . B o i l g e n t l y with a stream of Na flowing through the s o l u t i o n f o r 10 minutes a f t e r the temperature reaches 115°C. Cool with the N a s t i l l flowing) and sample up to 40 pg S i n an a l i q u o t to the b o i l i n g f l a s k . Adjust the N 2 flow to 75 ml m i n u t e - 1 per f l a s k . 4) Heat f o r 20 minutes so i t i s j u s t b a r e l y b o i l i n g . 5) Remove, and add 2.5 ml bismuth reagent and mix. Read immediately at 400 mp and compare against a standard. APPENDIX B4. DETERMINATION OF ACTIVE IRON IN FOLIAGE 1) Weigh 0.5 g subsample of oven-dried (70°C f o r 3 hours), ground f o l i a g e i n t o a 60 ml screw-capped p l a s t i c b o t t l e . 2) Add 10 ml 1 N HCL (reagent grade) i n d e m i n e r a l i z e d water to each sample. T i g h t l y cap the b o t t l e to prevent leakage. (70 samples can be prepared i n one r u n ) . 3) Shake the b o t t l e h o r i z o n t a l l y f o r 24 hours on a r e c i p r o c a t i n g shaker at room temperature. Have a blank and r e f e r e n c e samples f o r each s e t . 4) F i l t e r the e x t r a c t through Whatman # 41 f i l t e r paper and c o l l e c t the f i l t r a t e i n a 60 ml p l a s t i c b o t t l e . 5) Analyze f o r i r o n on atomic a b s o r p t i o n spectrophotometer. A n a l y s i s should be done w i t h i n 48 hours. 202 APPENDIX B5. DETERMINATION OF EXTRACTABLE ZINC 1) Weight 0.5 grains of a subsample of oven-dried (70°C f o r 3 hours), ground sample i n t o a 60 ml p l a s t i c b o t t l e . 2) Add 20 ml of 1.0 mM MES (2-(N-morpholino)ethanesulfonic a c i d ) (prepared i n d e m i n e r a l i z e d water) to each b o t t l e . 3) Shake for 5 hours h o r i z o n t a l l y on a r e c i p r o c a t i n g shaker at room temperature. Include a blank and a r e f e r e n c e sample with each s e t . 4) F i l t e r through Whatman #41 f i l t e r paper. 5) Analyze f o r Zn on atomic a b s o r p t i o n . 203 APPENDIX B6. WATER EXTRACTABLE MANGANESE FROM FOLIAGE 1) Weigh out 0.1 g of a subsample of oven-dried (at 70°C f o r 3 hours), ground f o l i a g e i n t o a 60 ml p l a s t i c b o t t l e . 2) Add 50 ml of dem i n e r a l i z e d water. 3) Shake f o r 1 hour h o r i z o n t a l l y on a r e c i p r o c a t i n g shaker at room temperature. Have a blank and ref e r e n c e sample f o r each s e t . 4) F i l t e r through Whatman #41 f i l t e r paper. 5) Determine Mn using atomic a b s o r p t i o n on the e x t r a c t s . 204 APPENDIX C. COMPARISON OF FOLIAR Zn, Mn, AND Fe LEVELS USING AA VERSUS ICP SAMPLE ZnAA ZnlCP MnAA MnlCP FeAA FelCP Ug g " 1 Given 1 30 21.7 65 82 .6 170 245. 2 28 22 .2 66 83.3 170 252 3 29 33.9 180 200 75 79.7 4 40 33.8 220 200 70 82 .8 5 58 53.5 240 207 220 361 6 58 53.2 240 210 260 315 7 13 6.8 620 633 35 7.6 8 185 178.6 1220 1344 50 16 .9 9 23 15 .6 540 510 30 12.5 10 22 14.9 520 507 35 14.4 11 27 21.3 2400 2506 35 0.5 12 60 58 1420 1521 32 5.2 13 113 107.6 880 846 30 7.3 14 114 107.5 840 845 32 12 15 18 9.9 1200 1198 35 15 .4 16 18 10.7 1100 1164 40 19.5 17 17 8.8 1080 1092 33 4.2 18 15 8.9 3880 3783 30 0.5 19 19 12.6 3120 3192 30 0.5 20 19 12.3 3100 3182 27 0.5 21 15 9.7 1040 1064 31 10.2 22 16 9.9 960 1076 32 14.2 23 11 5.6 600 583 32 9.9 24 23 16.3 900 937 30 7.2 25 24 16 .4 860 946 30 6.1 26 28 21.2 64 79.4 160 227 27 38 30.5 200 184 75 81 . 7 28 55 50.3 20 201 255 37 Sample Zn Mn Fe NBS 25 91 300 Tomato 62 238 690 1. Sample Numbers 1. 2, 26 N a t i o n a l Bureau of Standards (NBS) Orchard Leaves 3, 4, 27 Pine Reference 84-3 5, 6, 28 NBS Tomato Leaves 8 - 2 5 Hemlock F o l i a g e Samples 2. (U.S. Dept. of Commerce 1977) 3. (U.S. Dept. of Commerce 1976) 205 APPENDIX D. FORMULAS TO CONVERT AA VALUES TO THE CORRESPONDING ICP VALUES 1. Equation f o r Zn Equation f o r Mn y = 1.005x R a = 0.996 SE » 2.53 y = 1.017x R a = 0.998 SE = 63.57 - 6.075 Where x = the c o n c e n t r a t i o n measured using the AA y = the e q u i v a l e n t c o n c e n t r a t i o n on the ICP Data are from Appendix C. 206 APPENDIX E. RECOVERY OF ELEMENTS IN NATIONAL BUREAU OF STANDARDS SAMPLES USING ICP AND AA CalCP CaAA MglCP MgAA KICP KAA •eg g ORCHARD LEAVES 1.98 1.47 0.605 0.566 1.4 1.42 1.88 1.37 0.616 0.566 1.42 1.36 1.89 1.4 0.577 0.574 1.39 1.37 1.92 1.41 0.599 0.569 1.4 1.38 MEAN GIVEN 2.09 2.09 0.62 0.62 1.47 1.47 % RECOVERY 91.9 67.5 96.6 91.8 95.2 93.9 TOMATO LEAVES 2.94 2.05 0.644 0.602 4.18 4.2 2.87 2.18 0.653 0.61 4.18 3.92 2.7 2.01 0.625 0.604 4.05 3.84 MEAN GIVEN X RECOVERY 2.84 2.08 0.641 0.605 4.14 3.99 3 3 0.7 0.7 4.46 4.46 94.7 94.7 91.6 86.4 92.8 89.5 207 Appendix MnlCP E (concluded) MnAA ZnlCP ZnAA FelCP Fe AA A1ICP ug g ORCHARD LEAVES 82.6 65 21.7 30 245 170 241 83 .3 66 22 .2 28 252 170 233 79.4 64 21.2 28 22 7 160 218 MEAN 81.8 65 21.7 28.7 241 167 231 GIVEN 91 91 25 25 300 300 X RECOVERY 89.9 71.4 86.8 114.8 80.3 55 .7 TOMATO LEAVES 207 240 53.5 58 361 220 371 210 240 53.2 58 315 260 315 201 200 50 .3 55 370 255 389 MEAN 206 227 52.3 57 349 184 358 GIVEN 238 238 62 62 690 690 A l AA 180 180 160 173 240 290 255 262 % RECOVERY 86.6 95.4 84.4 91.9 50.6 26.7 208 APPENDIX F. PREPARATION AND ANALYSIS OF CELLULAR FRACTIONS FROM FOLIAGE 1) Weigh 20 g of oven-dried (70°C f o r 3 hours), ground sample of f o l i a g e i n t o 500 ml p l a s t i c b o t t l e s f o r the c e n t r i f u g e , and add 100 ml of a s u c r o s e - b u f f e r s o l u t i o n (0.5 M sucrose, 0.05 M T r i s - H C L ) . Shake to wet the p l a n t m a t e r i a l thoroughly. 2) A p r o g r e s s i v e s e p a r a t i o n was done using c e n t r i f u g a t i o n ; F r a c t i o n A 500 g f o r 5 minutes F r a c t i o n B 3000 g f o r 10 minutes F r a c t i o n C S e t t l e d by g r a v i t y F r a c t i o n D 15000 g f o r 30 minutes F r a c t i o n E Supernatant remaining The f r a c t i o n was the p e l l e t formed i n the bottom of the c e n t r i f u g e tube f o l l o w i n g c e n t r i f u g a t i o n of the supernatant. The remaining supernatant was decanted and r e c e n t r i f u g e d at the next highest speed. F r a c t i o n C formed f o l l o w i n g decanting the supernatant from which f r a c t i o n B had been formed and before c e n t r i f u g a t i o n at 15000 g. 3) F r a c t i o n s A to D were d r i e d i n an oven i n p o r c e l a i n c r u c i b l e s to a constant weight. F r a c t i o n E was st o r e d i n 60 ml p l a s t i c b o t t l e s i n the r e f r i g e r a t o r . 4) F r a c t i o n s A to D were d i g e s t e d using the Parkinson and A l l e n a c i d d i g e s t i o n . Because of the small amount of m a t e r i a l from f r a c t i o n D, the a c i d used f o r the d i g e s t i o n was added to the c r u c i b l e s l e f t o v e r n i g h t , and then t r a n s f e r r e d to the d i g e s t i o n tubes. 5) Zn and Mn were determined on the d i g e s t s and on the f r a c t i o n E supernatant using atomic a b s o r t i o n spectrophotometry. 209 APPENDIX G. FIXATION AND EMBEDDING PROCEDURE 1) Place t i s s u e i n a small v i a l to completely cover i t c o n t a i n i n g 2.5% glutarnaldehyde i n 0.1 M Na-cacodylate (pH 7.2-7.4). Leave i t f o r 3 hours. 2) The s o l u t i o n i s prepared by combining 10 ml of 25% g l u t a r a l d e h y d e , 50 ml of 0.2 M Na-cacodylate and 40 ml of de m i n e r a l i z e d water. 3) The t i s s u e i s than r i n s e d three times at 10-minute i n t e r v a l s i n 0.1 M Na-cacodylate (pH 7.2-7.4). This i s prepared by adding 40 ml of 0.2 M Na-cacodylate to 40 ml of d e m i n e r a l i z e d water. 4) The t i s s u e i s f i x e d f o r a second time f o r 1 hour i n a s o l u t i o n of 1% osmium t e t r o x i d e i n 0.1 M Na cac o d y l a t e (pH 7 .2-7.4) . Thi.!i i s p r e p a r e d by a d d i n g a 1:1:2 v o l u m e o f 2% aqua o s m i u m t « t r o x t d « s <lc*t« i n « r * l i y,*t<\ « « t ** r H I H I N a 5) The t i s s u e i s r i n s e d i n d e m i n e r a l i z e d water three times at 10 minute i n t e r v a l s . 6) The t i s s u e i s then taken through a dehydration s e r i e s using ethanol at 10-minute i n t e r v a l s i n the f o l l o w i n g c o n c e n t r a t i o n s of e t h a n o l ; 30%, 50%, 70%, 85%, 95%, 100%, 100%. 7) The t i s s u e i s then embedded i n the f o l l o w i n g s e r i e s ; f o r 10 minutes i n propylene oxide, twice, f o r 3 hours i n 3:1 mixture of propylene oxide and p l a s t i c , f o r 7-12 hours i n 1:1 propylene oxide and p l a s t i c , f o r 7-12 hours i n 1:3 propylene oxide and p l a s t i c , and then 7-12 hours i n 100% p l a s t i c . The t i s s u e i s then polymerized. 8) The p l a s t i c i s prepared by mixing 34.58 g of Epon Ep812 WPE #190, 13.0 g of DDSA (dodecenyl s u c c i n i c anhydride, 13.0 g of NMA nadic methyl anhydride, and 0.75 g of DMP -30. 210 APPENDIX H. THE MEHLICH 3 SOIL EXTRACTION METHOD 1) Weigh 5.0 g of s o i l samples i n t o 125 ml p l a s t i c b o t t l e s . Include 2 blanks and 2 r e f e r e n c e s s o i l s per run. 2) Have e x t r a c t i n g s o l u t i o n (M3) i n 4 L carboy with o u t l e t connected to t e f l o n tubing c l o s e d with c l i p . Set carboy on s t o o l on work bench. 3) C a l i b r a t e p l a s t i c graduated c y l i n d e r "to d e l i v e r " 50 ml. 4) Set up r e q u i r e d number of 60 ml p l a s t i c b o t t l e s with funnels and Whatman #541 X 15.0 cm f i l t e r paper. 5) Since e x t r a c t i o n time of samples should be p r e c i s e use a stopwatch on the bench. 6) F i l l graduated c y l i n d e r with M3 s o l u t i o n to the "to d e l i v e r " mark. 7) Pour i n t o 1st 125 ml b o t t l e , cap and put onto shaker at slow speed, s t a r t i n g the stopwatch. 8) R e f i l l the graduated c y l i n d e r and, when stopwatch gets to 50 seconds, pour M3 s o l u t i o n i n t o a 2nd 125 ml b o t t l e , cap and put onto shaker. 9) Continue t h i s at every 50 second reading ( i . e . at 60 second i n t e r v a l s ) of the stopwatch u n t i l the 6th b o t t l e i s put on the shaker, then immediately remove the 1st b o t t l e (which w i l l have shaken f o r 5 minutes) and pour through f i l t e r . Then t r a n s f e r the l a b e l to the 60 ml c o l l e c t i o n b o t t l e . 10) Continue adding 50 ml M3 s o l u t i o n to samples i n 125 ml b o t t l e s at 50 second readings on the stopwatch, p u t t i n g the b o t t l e immediately onto the shaker, and then immediately removing and f i l t e r i n g the sample which has shaken f o r 5 minutes. Thus there should always be 5 b o t t l e s shaking a f t e r the i n i t i a l s t a r t up, u n t i l the l a s t 5 samples, which should be removed from the shaker at 1 minute i n t e r v a l s , s t a r t i n g at the 58 second reading on the stopwatch. 11) Measure elements on ICP. 12) E x t r a c t s may be kept f o r a week, however d i l u t i o n s must be read w i t h i n 3 days. 211 Appendix H (concluded). E x t r a c t i n g S o l u t i o n Reagents: A l l should be ACS grade. Rl Ammonium n i t r a t e R2 Ammonium f l u o r i d e R3 A c e t i c a c i d , g l a c i a l 99.5%, 17.4 N R30 D i l u t e 200 ml g l a c i a l a c e t i c a c i d to 1000 ml with d e m i n e r a l i z e d water (DMHaO). R4 N i t r i c a c i d (HN0 3) 68-70%, 15.5 N R4D D i l u t e 20 ml cone. HN0s to 1000 ml with DMH20 R5 E t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA), fw 292.24 Stock Mehlich 3: Put about 120 ml DMHaO i n a 200 ml vo l u m e t r i c f l a s k . Add 27.78 g R2 and mix, then add 14.61 g EDTA, d i s s o l v e and make to 200 ml. Mix w e l l and immediately t r a n s f e r to a p l a s t i c b o t t l e . I t i s necessary to d i s s o l v e the R2 f i r s t and then add the EDTA to get the EDTA i n t o s o l u t i o n . E x t r a c t a n t : Use a p l a s t i c carboy with o u t l e t , c a l i b r a t e d to 4 L, and add about 3 L of DMHaO. Add 80.0 g Rl and d i s s o l v e . Add 16 ml Stock Mechlich 3 and mix. Measure with a p l a s t i c graduated c y l i n d e r . Add 230 ml R3D. Add 164 ml R4D. Make to 4 L with DMHaO and mix thoroughly. pH should be 2.5+0.1. 212 APPENDIX I. FOLIAR NUTRIENT GUIDELINES FOR THE INTERPRETATION OF NUTRITIONAL STATUS FOR HEMLOCK (FROM BALLARD AND CARTER (1986). I n t e r p r e t a t i o n N% P% K% 0 0 0 Very s e v e r e l y d e f i c i e n t S e v e r e l y d e f i c i e n t S l i g h t moderate d e f i c i e n c y Adequate Se v e r e l y d e f i c i e n t Moderate-severely d e f i c i e n t P o s s i b l e slight-moderate de f i c i e n c y L i t t l e , i f any d e f i c i e n c y No d e f i c i e n c y 1.05 0.08 0.35 1.3 0.1 0.45 1.45 0.15 0.75 Ca% Mg% 0 0 0.1 0.06 0.15 0.08 0.2 0.1 0.25 0.12 213 Appendix I (continued) ug S" 1 Mn Severe d e f i c i e n c y Probable d e f i c i e n c y P o s s i b l e d e f i c i e n c y or near d e f i c i e n c y No d e f i c i e n c y Fe He f i i ; i <̂  nr,y l i k e l y P n •- - a i h ~i t- (1 !• i <'. i (» 11 r. V Low to zero p r o b a b i l i t y of d e f i c i e n c y A c t i v e Fe D e f i c i e n c y l i k e l y D e f i c i e n c y u n l i k e l y Zn Probable d e f i c i e n c y P o s s i b l e d e f i c i e n c y No d e f i c i e n c y Cu Probable d e f i c i e n c y P o s s i b l e moderate d e f i c i e n c y P o s s i b l y somewhat d e f i c i e n t S l i g h t p o s s i b i l i t y of d e f i c i e n c y 0 4 15 25 0 25 50 0 30 0 10 15 0 1 2 2.6 4 No d e f i c i e n c y Appendix I (continued) B D e f i c i e n c y l i k e l y B p o s s i b l y d e f i c i e n t ; P o s s i b l e B probably not d e f i c i e n t If N<1.5%, then NID p o s s i b l e If N>1.5%, then HID u n l i k e l y No d e f i c i e n c y Appendix I (continued) N/P 0 No P d e f i c i e n c y ; NID* u n l i k e l y 6.11IN + 0.11 No P d e f i c i e n c y ; NID u n l i k e l y 12 P o s s i b l e P d e f i c i e n c y ; NID or NAD* p o s s i b l e 16 P d e f i c i e n c y P/Al 0 P/Al suggests P d e f i c i e n c y , unless P> 0.13% 3 No i n t e r p r e t a t i o n K/Ca 0 P o s s i b l e K d e f i c i e n c y 0.5 No i n t e r p r e t a t i o n 3.5 High K/Ca suggests d e s i r a b i l i t y of checking f o r p o s s i b l e Fe d e f i c i e n c y Ca/Mg Ca/Mg i s u n u s u a l l y low and may impair growth. If s o i l parent m a t e r i a l i s of u l t r a m a f i c o r i g i n , c o n s i d e r p o s s i b i l i t i e s of Mo d e f i c i e n c y and Ni and/or Cr t o x i c i t y . No i n t e r p r e t a t i o n a. NID = D e f i c i e n c y i n d u c i b l e by N f e r t i l i z a t i o n ; NAD = d e f i c i e n c y may be aggravated by N f e r t i l i z a t i o n . 0 0.8 b. N = N percent, dry mass b a s i s . 216 Appendix I (concluded) S If s u l f a t e - S i s not ev a l u a t e d : data suggests a c t u a l or i n d u c i b l e S d e f i c i e n c y . If s u l f a t e - S < 0.01%, p o s s i b l e S d e f i c i e n c y and p o s s i b l e NAD 1. If s u l f a t e - S >0.01%, no S d e f i c i e n c y but NID 1 p o s s i b l e . P o s s i b l e S d e f i c i e n c y and If s u l f a t e - S < 0.01%, NID- p o s s i b l e . I f H II I. f H I*.«—S > 0 .01%, NID u n l i k e l y . S d e f i c i e n c y and NID are both u n l i k e l y . No S d e f i c i e n c y ; NID u n l i k e l y . N/S (Not i n t e r p r e t e d where t o t a l S exceeds 0.14%) No S d e f i c i e n c y ; NID u n l i k e l y . No S d e f i c i e n c y ; NID p o s s i b l e . P o s s i b l e S d e f i c i e n c y . S d e f i c i e n c y . 0.00 % 0.12 % 0.14 % 0.16 % 0 4.2N + 4.94* 13.6 14.6 S u l f a t e - S (The f o l l o w i n g a p p l i e s only where t o t a l S has not been evaluated) 0.000 % Ac t u a l or i n d u c i b l e S d e f i c i e n c y ; NAD or NID l i k e l y . 0.008 % No S d e f i c i e n c y ; but NID p o s s i b l e . 0.020 % No S d e f i c i e n c y ; but NID u n l i k e l y . 0.040 % Very high; p o s s i b l y N d e f i c i e n t . a. NID = D e f i c i e n c y i n d u c i b l e by N f e r t i l i z a t i o n ; NAD = d e f i c i e n c y may be aggravated by N f e r t i l i z a t i o n . b. N = N percent, dry mass b a s i s . 217 APPENDIX J . CONCENTRATIONS OF ZINC HEMLOCK FOLIAGE. AND MANGANESE IN THE CELLULAR FRACTIONS OF MEANS SAMPLE ZNA MNA ZNB MNB ZNC •ug s~x- 1 5.3 603 5.3 447 7.7 2 6.3 1460 7.3 1277 8.0 3 41.0 528 34.0 430 45.3 4 4.0 677 4.3 657 6.4 5 5.3 114 7.3 95 7.3 6 9.3 160 9 . 7 153 9.9 7 8.3 15 8.0 15 10.6 STANDARD DEVIATION 1 1.5 62 2 1.2 32 3 14.8 82 4 0.0 49 5 1.5 3 6 1.2 31 7 1.2 3 1.5 36 1.2 0.6 56 2.0 11.3 75 15.9 0.6 125 0.7 2.1 10 0.6 0.6 18 0.8 2.0 4 1.6 MINIMUM 1 4.0 545 2 5.0 1437 3 24.0 452 4 4.0 622 5 4.0 111 6 8.0 127 7 7.0 13 4.0 407 7.0 7.0 1227 6.0 21.0 387 27.0 4.0 537 5.7 5.0 83 7.0 9.0 137 9.0 6.0 12 8.8 MAXIMUM 1 7.0 668 2 7.0 1497 3 51.0 615 4 4.0 717 5 7.0 117 6 10.0 187 7 9.0 18 7.0 477 9.0 8.0 1337 10.0 41.0 517 55.0 5.0 787 7.0 9.0 102 8.0 10.0 172 10.4 10.0 19 11.7 Appendix J (concluded) SAMPLE MNC ZND MND ZNE MNE 1 740 46.4 661 6.1 1300 2 1717 74.4 1692 7.4 2513 3 602 131.3 668 68.7 4 844 56.4 824 5 .4 1280 5 130 110.9 242 8.1 205 6 198 48.5 232 7.9 315 7 19 • 42.6 39 1.0 9 MNC ZND MND ZNE MNE 1 91 35 .0 123 0.5 96 2 17 35.7 141 0.4 153 3 90 26.6 113 20.5 4 64 25 .3 126 0.3 46 5 6 20.9 35 0.1 15 6 12 21.2 58 0.4 28 7 10 44.2 34 0.1 1 MNC ZND MND ZNE MNE 1 657 16.9 525 5.8 1230 2 1707 50.6 1532 7.1 2380 3 527 108.4 580 45 .0 4 776 31.2 687 5.0 1240 5 127 87.7 217 8.0 190 6 185 26.1 183 7.6 285 7 8.8 0.0 0 0.9 9 MNC ZND MND ZNE MNE 1 837 85 .1 766 6.7 1410 2 1737 115 .4 1800 7.9 2680 3 702 160.5 795 81.5 4 902 81.8 936 5.6 1330 5 137 128.2 282 8.3 220 6 207 68.2 295 8.4 340 7 28.1 88.2 59 1.0 10 Sample 1. Hemlock, s i t e 5, c o n t r o l , 1986. 2. Hemlock, s i t e 5, treatment 6, 1986. 3. Hemlock, s i t e 5, treatment 15, 1986. 4. Hemlock, s i t e 5, 1987. 5. D o u g l a s - f i r , s i t e 5, 1987. 6. Amabilis f i r , s i t e 5, 1987. 7. Yellow cedar, s i t e 5, 1987. 219 A P P E N D I X K . S C A T T E R PLOTS OF HEIGHT INCREMENT VERSUS F O L I A R MASS PER SHOOT. 8 0 -| U C D 0 0 CJ) 6 0 H c <D 40H CD c 20 J Z .CP y = 291.Ox - 440.Ox R = 0.38 Q I i i i i i i i i i I i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | 0.0 0.1 0.2 0.3 0.4 0.5 Foliar Mass Per Shoot 1986 (g) A p p e n d i x K . 1 . S c a t t e r p l o t o f t h e 1986 f o l i a r mass p e r s h o o t t h e f o r 1986 s i t e h e i g h t i n c r e m e n t v e r s u s 2 . 220 80q E : o DO 0.2 0.4 0.6 0.8 Foliar Mass Per Shoot 1986 (g) A p p e n d i x K . 2 . S c a t t e r p l o t o f t h e 1986 h e i g h t i n c r e m e n t v e r s u s t h e 1986 f o l i a r mass p e r s h o o t f o r s i t e 3 . 221 1 0 0 n y = 160.Ox - 71.Ox 0 0 0 . 4 0 . 8 1 . 2 1.6 Foliar Mass Per Shoot 1986 (g) A p p e n d i x K . 3 . S c a t t e r p l o t o f t h e 1 9 8 6 f o l i a r mass p e r s h o o t t h e f o r 1986 s i t e h e i g h t i n c r e m e n t v e r s u s 4 . 222 160.0 n E : o Foliar Mass Per Shoot 1987 (g) A p p e n d i x K . 4 . S c a t t e r p l o t o f t h e 1 9 8 7 h e i g h t i n c r e m e n t v e r s u s t h e 1987 f o l i a r mass p e r s h o o t f o r s i t e 4 . 223 80.0 -| A p p e n d i x K . 5 . S c a t t e r p l o t o f t h e 1986 h e i g h t i n c r e m e n t v e r s u s t h e 1986 f o l i a r mass p e r s h o o t f o r s i t e 5 . 224 120.0 -i '100.0 -3 oo 80.0 H c 0 60 .0 CD ^ 40 .0 i ^ 20 .0 ^ CD 0.0 y = 206.Ox - 124.Ox R = 0.49 0.0 i 1 1 u i i i ! i i 1 1 1 1 1 1 i I I I i i i i n i 11 1 1 i 1 1 i i i i i M 1 1 i u 111 11 i i i i i n r 0.2 0 .4 0.6 0.8 1.0 1.2 Foliar Mass Per Shoot 1987 (g) A p p e n d i x K . 6 . S c a t t e r p l o t o f t h e 1987 h e i g h t i n c r e m e n t v e r s u s t h e 1987 f o l i a r mass p e r s h o o t f o r s i t e 5 . APPENDIX L. FOLIAR NUTRIENT DATA. MEANS l-85 ,--85 a-85 3 TRT ZN MN --ug g - 1 1 10.9 1284 2 14.2 1131 3 15 .1 3008 4 58 .1 1133 5 149 .7 1180 6 9.4 1354 7 10.5 1175 8 7.9 1129 9 9.6 1008 10 9.2 1082 11 7.2 1022 12 7.6 1002 STANDARD DEVIATION !T ZN MN 1 5.6 330 2 9.5 367 3 5.8 1298 4 15 .1 292 5 46 .8 327 6 2.5 416 7 3.4 325 8 2.1 352 9 4.0 388 10 2.4 426 11 1 . 7 321 12 1.9 306 P FE CU B C g g p g g 0.199 37.8 2.1 13.6 0 .225 40.1 2.4 13 .3 0.181 41.5 2.9 14.4 0.177 40 .2 2.5 14.5 0.174 41.6 2.1 13 .5 0 .192 40 .9 2.4 15 .4 0 .176 43.3 2.4 13.3 0.185 42.2 2.2 13.3 0.191 41.4 2.2 16.5 0.187 39.5 2.3 13.6 0.168 40.3 2.2 13.8 0.185 38 .9 2.4 14.2 P FE CU B 0 .043 5.4 0.6 3.6 0 .057 5.3 0.6 4.4 0 .060 5.4 0 . 7 5.3 0.020 3.7 0.4 3.5 0 .041 4.9 0.3 2.9 0.032 6.2 0.6 4.4 0 .025 3.5 0 . 7 2.6 0 .034 11.3 0.5 4.3 0.038 5.9 0.6 9.0 0 .019 4.2 0.6 3.2 0.024 4.1 0.8 4.8 0 .034 5.2 0.7 6.3 1. Year of f e r t i l i z e r treatment. 2. Year of f o l i a g e c o l l e c t i o n . 3. Year i n which f o l i a g e was formed. Appendix L ( c o n t i n u e d ) . NUMBER OF CASES TRT ZN MN 1 10 10 2 9 9 3 8 8 4 8 8 5 7 7 6 10 10 7 8 8 8 9 9 9 10 10 10 9 9 11 10 10 12 10 10 MEAN TRT SOS TOTS FLWTBR Mg g _ 1 eg g ~ l g 1 0.236 2 0 .221 3 0.360 4 0 .180 5 0.241 6 0 .175 7 342 0.146 0 .202 8 509 0.160 0 .208 9 671 0.165 0 .239 10 851 0.190 0.219 11 460 0.131 0.188 12 401 0.124 0 .283 P FE CU 10 10 10 9 9 9 8 8 8 8 8 8 7 7 7 10 10 10 8 8 8 9 9 9 10 10 10 9 9 9 10 10 10 10 10 10 BI1 HTI1 HTI2 0.80 0.73 0.91 0.64 0.60 1.18 0.84 0.78 2.02 0.59 0.65 1.15 0.80 0.51 2.54 0.63 0.69 1.26 0.77 0.69 0.97 0.76 0.73 0.83 0.74 0.72 1.08 0.75 0.61 0.89 0.71 0.56 0.77 0.81 0.62 0.79 Appendix L ( c o n t i n u e d ) . STANDARD DEVIATION TRT SOS TOTS FLWTBR 1 0.121 2 0.099 3 0 .246 4 0.042 5 0 .154 6 0.059 7 204 0.024 0.062 8 182 0.012 0.076 9 264 0.019 0 .133 10 112 0.044 0.163 11 130 0.007 0 .050 12 81 0 .013 0.127 NUMBER OF CASES TRT SOS TOTS FLWTBR 1 0 0 10 2 0 0 9 3 0 0 8 4 0 0 8 5 0 0 7 6 0 0 10 7 8 8 8 8 8 8 9 9 10 10 10 10 9 9 9 11 10 10 10 12 - 10 10 10 BI1 HTI1 HTI2 0.29 0.31 0.38 0.15 ; 0.23 0.47 0.32 0.35 1.32 0.14 0.23 0.64 0.28 0.19 1.14 0.17 0.21 0.41 0.17 0.20 0.73 0.16 0.27 0.30 0.22 0.57 0.56 0.25 0.24 0.35 0.18 0.21 0.18 0.17 0.22 0.50 BI1 HTI1 HTI2 10 7 7 9 8 8 8 8 8 8 7 7 7 7 7 10 9 9 8 8 8 9 9 9 10 10 10 9 8 8 10 9 9 10 10 10 228 Appendix L ( c o n t i n u e d ) . 1-85-86-86 HE AN TRT ZN HN P FE CU PS g-X eg S~ l pg g " 1 1 13.3 1083 0.189 61.2 3.0 2 17.2 915 0.228 67.8 3.6 3 11.0 2971 0.169 62 .9 4.3 4 13.4 1247 0.203 75 .5 3.6 5 15 .2 1142 0.192 70.6 3.3 6 10 .6 1177 0 .200 71.0 3.7 7 11.5 1231 0.184 59.4 2.7 8 11.1 1160 0.184 70.2 2.3 9 9.9 856 0.184 53.6 2.2 10 11.4 914 0.193 58.7 2.4 11 8.8 992 0.197 70.8 3.1 12 9.7 1139 0.207 77.8 3.2 STANDARD DEVIATION TRT ZN HN P FE CU 1 3.9 446 0.041 20 . 7 1.0 2 10.3 285 0.041 33.8 0.7 3 4.6 1278 0 .039 18.4 2 .1 4 2.2 656 0.029 14.0 0.7 5 5.2 382 0.071 29.8 0.8 6 1.8 488 0 .028 32 .5 0.9 7 3.5 352 0 .024 15 .8 0.6 8 3.5 477 0.050 28.1 0.7 9 2.4 407 0 .035 11.4 0 . 7 10 1.5 378 0.034 17.6 1.2 11 2.3 200 0 .040 19 .4 0.6 12 3.2 395 0.045 16 .2 0.9 229 Appendix L ( c o n t i n u e d ) . NUMBER OF CASES TRT ZN MN P FE CU 1 7 7 7 7 7 2 9 9 9 9 9 3 8 8 8 8 8 4 7 7 7 7 7 5 9 9 9 9 9 6 8 8 8 8 8 7 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 10 7 7 7 7 7 11 9 9 9 8 8 12 9 9 9 9 9 MEAN TRT B TS FLWTBR BI2 ug g - 1 eg g - 1- g 1 21.8 0.248 1.26 2 23.8 0.221 1.59 3 19.6 0.348 1.54 4 24.5 0.264 1.22 5 22.9 0.206 1.61 6 22.5 0.193 1.06 7 20.8 0.151 0.202 1.10 8 22.5 0.153 0.140 0.87 9 22.8 0.156 0.213 0.99 10 22.3 0.157 0.343 1.36 11 23.7 0.143 0.159 0.92 12 23.2 0.150 0.195 0.84 Appendix L ( c o n t i n u e d ) . STANDARD DEVIATION TRT B TS FLWTBR 1 6.2 0.206 2 4.1 0.120 3 6.1 0.192 4 6.4 0.193 5 3.6 0.149 6 6.5 0.141 7 4.0 0.020 0.117 8 7.2 0.021 0.056 9 10.4 0.018 0.131 10 4.0 0.020 0.231 11 6.6 0.015 0.061 12 8.3 0.031 0.131 NUMBER OF CASES TRT B TS FLWTBR 1 7 0 24 2 9 0 27 3 8 0 24 4 7 0 21 5 9 0 27 6 8 0 24 7 8 8 24 8 9 9 18 9 9 9 27 10 7 7 18 11 8 7 27 12 9 8 30 BI2 1.02 1.92 0.69 0.51 0.71 0.32 0.52 0.23 0 .33 0 .88 0 .24 0.31 BI2 24 27 24 21 27 24 24 18 27 18 27 30 231 Appendix L ( c o n t i n u e d ) . 2-85-85-85 MEAN TRT ZN MN P FE CU Mg g - 1 eg g - 1- jig g - t 1 6.9 903 0.181 35 .7 2.8 2 7.9 1403 0 .126 37.7 2.9 3 7.2 3142 0.111 36.5 3.1 4 58.8 164.8 0.188 40.0 3.2 5 75 .0 1230 0.124 35 .7 3.6 6 9.0 1380 0.163 44.6 3.3 7 6.7 1167 0.141 35.7 3.0 8 5.8 1140 0.163 37.1 2.8 9 8.3 1343 0.136 35 .7 2.7 10 8.5 1268 0.167 40.8 3.0 11 7.0 1030 0.136 40.5 3.1 12 6.9 1449 0 . 126 38 .3 3.1 STANDARD DEVIATION TRT ZN MN 1 2.8 393 2 1.6 489 3 3.3 1470 4 21.8 548 5 99.0„. 99 6 3.0 328 7 2.3 794 8 2.8 667 9 6.2 401 10 2.3 572 11 2.4 172 12 2.5 655 P FE CU 0.050 4.6 0.5 0.044 6.5 0.8 0.021 4.6 0.4 0.056 3.0 0.4 0.042 0.053 7.4 0.9 0.037 7.1 0.4 0.046 5.4 0.8 0.036 12.9 0.5 0.060 6.1 0.3 0.027 2.1 1.1 0.040 4.0 0.8 232 Appendix L ( c o n t i n u e d ) . NUMBER OF CASES TRT ZN MN P FE CU 1 7 7 7 7 7 2 8 8 8 7 7 3 9 9 9 9 9 4 5 5 5 5 5 5 2 2 2 1 1 6 6 6 6 4 4 7 3 3 3 3 3 8 5 5 5 5 5 9 8 8 8 7 7 10 8 8 8 7 7 11 4 4 4 3 3 12 9 9 9 7 7 [EAN B TOTS SOS FLWTBR BI1 HTI2 HTI1 U g 6" 1 e g g ~ l u g g " 1 S 13.9 0.143 0.76 1.29 1.05 17.5 0.129 0 . 76 1 .92 0 . 77 17.3 0.145 0.82 1.82 0 .90 16 .6 0 .153 0.86 1.57 0.72 18.2 0.077 0.59 1.85 0.55 17.3 0.136 0.79 1.32 0.83 15 .7 0.119 407 0.107 0.74 1.42 0.75 18.6 0 .151 635 0.118 0 . 75 1.57 0.91 19.6 0.141 585 0.144 0.87 1.50 0.85 17.1 0.160 837 0.168 0.72 1.35 0.77 17.1 0.624 505 0.136 0.73 1 .38 0.63 18 .9 0.273 621 0.143 0 . 71 1 .20 0.94 Appendix L ( c o n t i n u e d ) . STANDARD DEVIATION B TOTS SOS FLWTBR BI1 HTI2 HTI1 3 . 7 0 .048 0.16 0.58 0 .35 5.4 0.040 0.14 0.96 0.35 4.0 0 .056 0.15 0.51 0 .12 3.2 0.034 0.22 0.85 0.32 3.9 0.041 1.62 0.16 8.7 0.048 0.18 0.66 0.33 2.4 0.027 51 0.019 0.01 0.41 0.28 3.4 0.023 305 0.042 0.21 0.20 0.54 2.7 0 .012 149 0 .037 0.28 0 .39 0.18 5.7 0.019 109 0.059 0.11 0.68 0.39 8.1 0 .889 370 0.054 0.08 0 . 73 0.14 4.2 0.395 135 0.049 0.15 0.45 0.34 IHBER OF CASES B TOTS SOS FLWTBR BI1 HTI2 HTI1 7 0 0 7 7 6 6 7 0 0 8 8 7 7 9 0 0 9 9 7 7 5 0 0 5 5 7 7 2 0 0 2 1 5 6 6 0 0 6 6 4 4 3 2 2 3 3 3 3 5 5 5 5 5 5 5 7 8 8 8 8 6 6 8 8 7 8 8 4 5 4 3 3 4 4 4 4 8 8 8 9 9_ 7 7 234 Appendix L ( c o n t i n u e d ) . 2-85-86-86 MEAN TRT ZN MN P FE CU Ug g-X eg S~x pg g-* 1 13.3 1323.8 0 .145 53.7 2.4 2 13.6 1399.3 0.148 42.9 2.3 3 12.4 3875.1 0.128 39.3 2.5 4 11.5 1526.7 0.153 55 .6 3.1 5 19 .6 1140.8 0.130 52.8 2.7 6 12.7 1327.8 0.118 51.4 2.8 7 8.8 1727.5 0.140 31.4 2.6 8 11.4 1180 .8 0 .152 35.1 2.7 9 11.4 1401.4 0.136 33.8 2.6 10 13.0 2088 .0 0 .132 32.5 2.9 11 10.1 1249.3 0.125 57.1 2.9 12 11.1 1931.8 0.126 41.4 2.8 1ARD DEVIATION TRT ZN MN P FE CU 1 3.8 289 .3 0 .064 18.0 0.4 2 2.7 433 .9 0.042 14.4 0.9 3 5.6 1428.5 0 .042 13.2 0.6 4 5.6 1031.0 0.035 10.3 1.0 5 8.8 433.6 0.055 9.6 0 . 7 6 3.7 370.9 0 .024 23.3 0.5 7 3.4 1025 .1 0.028 7.7 0.9 8 2.7 601.0 0.042 5.4 0.6 9 6.8 359.5 0.033 8.0 0 . 7 10 5.5 985 .4 0.027 7.1 0.7 11 4.2 357.4 0.029 14.3 0.9 12 4.8 589.0 0.029 7.8 0.4 235 Appendix L ( c o n t i n u e d ) . NUMBER OF CASES TRT ZN 1 6 2 6 3 9 4 7 5 5 6 5 7 4 8 6 9 7 10 5 11 4 12 8 MN 6 6 9 7 5 5 4 6 7 5 4 8 P 6 6 9 7 5 5 4 6 7 5 4 8 FE 6 6 9 7 5 5 4 6 7 5 4 8 CU 6 6 9 7 5 5 4 6 7 5 4 8 MEAN TRT B TOTS FLWTBR BI2 Ug g ~ l eg g " 1 g 1 28.8 0.140 1.31 2 25 .8 0.196 1.58 3 33.0 0.181 1.42 4 28 . 7 0.158 1.58 5 26.5 0.131 1.34 6 30.8 0.190 1 .06 7 37.1 0.147 0.123 1 .23 8 22 .9 0 .156 0.163 1 .08 9 30.0 0.153 0.140 1 .28 10 31.3 0.162 0.156 1.14 11 31.0 0.140 0.163 1.47 12 30.0 0.148 0.186 1 .02 236 Appendix L ( c o n t i n u e d ) . STANDARD DEVIATION TRT B TOTS FLWTBR BI2 I 7.5 0 .047 0.48 2 7.6 0.119 0.53 3 9.4 0 .096 0.55 4 5.0 0.068 0.68 5 6 . 7 0.092 0.51 6 4.1 0.104 0.25 7 13 .1 0.012 0.039 0 .45 8 4.9 0.019 0.113 0.51 9 3.8 0 .008 0.057 0.40 10 6.1 0.019 0.147 0.53 11 10.2 0 .008 0.074 0.76 12 5.0 0.012 0.076 0 .33 NUMBER OF CASES TRT B TOTS FLWTBR BI2 1 6 0 18 18 2 6 0 18 18 3 9 0 27 27 4 7 0 18 18 5 5 0 15 15 6 5 0 14 14 7 4 3 12 12 8 6 5 18 15 9 7 6 24 24 10 5 5 15 15 11 4 4 12 12 12 8 6 24 24 Appendix L ( c o n t i n u e d ) . 3-85-86-86 MEAN TRT ZN MN P B TOTS pg g - i eg g " 1 pg g - x eg g" 1 11.1 1502 0.174 16.2 2 17.7 1638 0 .193 25 . 7 3 15 .5 2427 0 .187 22.5 4 21.3 2019 0 .220 27.8 5 19.1 1793 0.178 21.7 6 15 .4 1899 0.173 27.8 7 14.4 2197 0.230 28 .1 0.158 8 17.4 2268 0.170 28.7 0.140 9 13.1 1245 0.185 28.2 0.145 10 12 .8 1212 0 .173 26.6 0.140 11 17.9 1853 0.180 22.7 0.143 12 11 . 7 1505 0 . 188 23.6 0 .140 STANDARD DEVIATION TRT ZN MN P B TOTS 1 1.9 455 0.026 3.2 2 10.0 648 0.028 5.4 3 2 .4 441 0.010 4.6 4 2.4 994 0.036 8.7 5 8.3 775 0.049 8.7 6 6.7 839 0.039 10.2 7 3.7 331 0.047 5 . 7 0.023 8 3.4 1037 0.038 12.2 0.029 9 3.5 280 0.021 4.5 0 .021 10 4.3 795 0.022 9.1 0.024 11 5.5 547 0 .026 4.8 0.047 12 1.7 287 0.033 9.2 0.023 Appendix L (continued) NUMBER OF CASES TRT ZN 1 7 2 7 3 7 4 3 5 8 6 8 7 8 8 8 9 4 10 4 11 8 12 6 MN 7 7 7 3 8 8 8 8 4 4 8 6 P 7 7 7 3 8 8 8 8 4 4 8 6 B 7 6 7 3 8 8 8 8 4 4 8 6 TOTS 0 0 0 0 0 0 8 8 4 4 8 6 MEAN TRT FLWTBR 8 BI2 BI1 HTI2 HTI1 1 2 3 4 5 6 7 8 9 10 11 12 0 0 0 0 0 0 0 0 .241 ,303 ,308 255 ,193 214 ,171 172 0.252 0.303 0.315 0.262 0.90 0.87 0.99 1.02 1.02 1 .00 0.86 0 . 79 1.02 0.92 1.05 0.95 0 .94 1.06 1.09 1 .18 0.78 0.93 0.86 0.82 0.93 1.09 0.90 0.88 1 1 1 0 1 1 0 1 0.97 1.17 1.20 1.28 04 16 05 99 10 07 79 09 1.01 1.00 1.07 1.15 0.92 1.07 1.00 0.84 0 .80 1 .18 0.94 0.83 Appendix L ( c o n t i n u e d ) . STANDARD DEVIATION TRT FLWTBR BI2 1 0 .106 0 .28 2 0.248 0.23 3 0.175 0.23 4 0.125 0.49 5 0.081 0.32 6 0.087 0.50 7 0 .089 0.37 8 0.105 0.30 9 0 .072 0.57 10 0.360 0.26 11 0.210 0 .35 12 0.085 0.26 NUMBER OF CASES TRT FLWTBR Bl: 1 21 21 2 21 21 3 21 21 4 12 12 5 23 23 6 22 22 7 27 27 8 26 26 9 12 12 10 15 15 11 27 27 12 24 24 BI1 HTI2 HTI1 0 .35 0.58 0.28 0.33 0.48 0.29 0.40 0.27 0 .36 0.20 0.21 0.41 0.38 0.37 0.44 0.34 0.54 0.22 0.27 0.22 0.45 0.23 0.30 0.45 0.24 0.07 0.23 0.37 0.47 0.37 0.42 0.46 0.36 0.26 0.53 0.38 BI1 HTI2 HTI 21 8 8 21 6 6 21 8 8 12 3 3 23 8 8 22 6 6 24 9 9 26 9 9 12 4 4 15 5 5 24 9 9 24 8 8 Appendix L ( c o n t i n u e d ) . 4-86-86-86 MEAN TRT ZN MN P6 g ~ l ~ 1 10.9 1469 2 8.7 1112 3 11.8 1098 4 9.2 2026 5 11.4 2802 6 9.1 3206 7 8.8 1012 8 9.6 1360 9 8.1 1168 10 11.3 1639 11 10.7 1647 12 11.8 1175 13 18.3 1448 14 26 .2 1384 15 48.6 1536 16 9.1 1208 17 7.7 1169 18 8.2 1464 19 9.4 1389 20 6.4 986 21 10.4 1411 22 7.8 1433 23 17.2 1343 24 9.2 1432 25 8.7 1428 N p -1 MG cg g 1.07 0 .170 0 .136 0.99 0 .175 0 .125 0.97 0 .153 0 .118 1.18 0 . 120 0 . 140 1.30 0 .125 0 . 140 1.33 0 . 138 0 .118 1.05 0 .160 0 .135 1 .04 0 .178 0 .140 0.94 0 .150 0 .128 1.09 0 .184 0 .136 0.98 0 .178 0 .158 1 .16 0 .134 0 .126 1.10 0 .163 0 .130 1.04 0 . 155 0 . 123 1.06 0 .178 0 .148 0 .99 0 . 160 0 . 126 1.01 0 .148 0 .120 0.97 0 .155 0 . 143 1.13 0 .148 0 .123 0.91 0 .118 0 .110 1.09 0 .162 0 .124 0.97 0 .135 0 .138 1.67 0 .160 0 .118 1.12 0 .136 0 . 130 1.00 0 .197 0 .140 Appendix L (continued) STANDARD DEVIATION TRT ZN MN PS s~ r 1 4.5 445 2 2.3 378 3 4.6 264 4 1.7 727 5 4.5 710 6 1.5 1341 7 2.9 257 8 2.9 432 9 2.6 456 10 4.3 597 11 6.0 604 12 3.6 214 13 6.9 247 14 10.4 283 15 25 .1 724 16 2.0 415 17 3.3 287 18 3.3 550 19 3.1 112 20 2.1 239 21 4.3 613 22 2 . 1 310 23 6.2 400 24 2.6 410 25 1.9 360 N P MG -eg g 0 .28 0 .010 0 .025 0 .18 0 .053 0 .024 0 .12 0 .051 0 .022 0 .07 0 .023 0 .016 0 .13 0 .035 0 .014 0 .18 0 .015 0 .029 0 .35 0 .042 0 .029 0 .29 0 .036 0 .008 0 .13 0 .042 0 .011 0 .23 0 .040 0 .034 0 .25 0 .050 0 .017 0 .25 0 .038 0 .034 0 .20 0 .057 0 .029 0 .09 0 .047 0 .015 0 .32 0 .073 0 .051 0 .20 0 .034 0 .023 0 .24 0 .031 0 .021 0 .35 0 .013 0 .010 0 .17 0 .039 0 .017 0 .17 0 .025 0 .014 0 .23 0 .040 0 .015 0 .13 0 .006 0 .022 0 .26 0 .014 0 .022 0 .19 0 .030 0 .025 0 .06 0 .071 0 .062 242 Appendix L (continued) NUMBER OF CASES TRT ZN MN N P MG 1 5 5 5 5 5 2 4 4 4 4 4 3 4 4 4 4 4 4 5 5 5 5 5 5 4 4 4 4 4 6 4 4 4 4 4 7 4 4 4 4 4 8 4 4 4 4 4 9 5 5 5 5 5 10 5 5 5 5 5 11 4 4 4 4 4 12 5 5 5 5 5 13 4 4 4 4 4 14 4 4 4 4 4 15 4 4 4 4 4 16 5 5 5 5 5 17 5 5 5 5 5 18 4 4 4 4 4 19 4 4 4 4 4 20 4 4 4 4 4 21 5 5 5 5 5 22 4 4 4 4 4 23 4 4 4 4 4 24 5 5 5 5 5 25 3 3 3 3 3 Appendix L (continued) MEAN FE CU B AFE FLWTBR BI2 — l. 8 --ug g 30.7 3.1 26.2 37.0 0.193 0.91 24.1 3.0 23 .1 32.3 0 .253 0.91 38.4 3.1 24.1 28.8 0.225 1 .18 25 .0 3.6 22.5 31.8 0.270 1.03 24.1 3.7 23.4 31.9 0.352 1.15 33.9 3 . 7 23.0 36.3 0.363 1.18 30.9 2.5 36.7 30.8 0.229 0.92 27.4 2.7 36 .9 30.8 0 .308 0 .92 35.7 2.3 38.8 31.8 0.191 0.85 32 .1 2.9 42 .2 32.5 0.271 0 .83 32 .1 2.5 89.5 32 .6 0.197 0.85 33 .6 2.6 79.0 32 .0 0.287 0.90 29.5 3.7 24.3 34.9 0.263 0.98 25 .9 3.8 24.0 35 .0 0.299 0.84 31.2 3.1 25.1 33.0 0.171 0.79 25 .0 3.9 25.7 30 .2 0 .286 0.83 32 .1 4.0 22 .8 35.2 0.211 0.82 34.8 2.6 23.8 32 .0 0 .216 0 .95 35 .7 2.7 21.8 30.8 0.267 0.80 28.6 2.3 22 . 7 27.5 0.136 1.04 34.3 2.9 26.8 30 .6 0.317 0.94 33.0 2.9 22.9 30.8 0.243 0.94 38.4 3.5 33.2 37.2 0.804 1.23 32.1 2.6 26 .0 30.2 0.198 0.78 25.0 , 3.2 24.5 32 .0 0.200 0.76 Appendix L (continued) STANDARD DEVIATION FE CU B AFE FLWTBR BI2 8 pg g 12 .8 1.0 4.1 9.3 0.100 0.39 8.9 0.6 7.0 3.9 0.170 0.21 29.9 0.4 6.1 5.8 0.083 1.13 5.0 0.4 6.3 3.8 0.096 0.26 8.4 0.9 2.8 5.5 0.388 0.38 8.5 0.4 2.5 3.3 0 .284 0.37 4.1 0.6 6.7 4.2 0.112 0.27 2 .1 0.7 15 .2 1. 7 0.2 76 0.31 11.6 0.5 7.5 2.0 0.080 0.34 5.6 0.3 11.0 4.4 0.178 0.27 12.4 0.8 30.7 6.0 0.058 0.28 6.0 0.6 21.6 4.1 0.239 0.32 10.3 0.7 2.4 7.1 0.156 0.47 5.4 0.6 4.9 5.7 0.082 0.39 4.5 0.8 5.1 5.4 0.136 0.35 5.6 0.9 3.7 4.0 0.231 0.29 9.2 1.2 9.6 8.4 0.073 0.24 3.4 0.5 6.8 1.6 0 .231 0.46 9.2 0.5 2.5 5.5 0.071 0.17 5.8 0.8 3.2 5.8 0.063 0.37 7.4 0.5 2.7 6.2 0.239 0.29 6.1 0.6 3.7 3.5 0 .062 0.46 1.8 0.6 4.1 2.4 0.646 0.48 5.6 0.7' 2.9 1.8 0 .120 0 .28 1.8 0.127 0.22 Appendix L (continued) NUMBER OF CASES FE CU B AFE FLWTBR BI2 5 5 5 5 5 30 4 4 4 4 4 27 4 4 4 4 4 27 5 5 5 5 5 30 4 4 4 4 4 24 4 4 4 4 4 26 3 3 3 3 4 27 3 3 3 3 4 21 5 5 5 5 5 26 5 5 5 5 5 30 4 4 4 4 4 30 5 5 5 5 5 24 4 4 4 4 4 30 4 4 4 4 4 21 4 4 4 4 4 25 5 5 5 5 5 27 4 4 4 4 5 28 4 4 4 4 4 27 4 4 4 4 4 30 4 4 4 4 4 27 5 5 5 5 5 30 4 4 4 4 4 27 4 4 4 4 4 30 5 5 5 5 5 30 1 1 3 1 3 24 Appendix L (continued) 4-86-87-87 MEAN TRT ZN MN Pg 8" 1 1 11.7 1719 2 8.5 1302 3 11.2 1465 4 10.1 2980 5 9.2 3460 6 9.7 3737 7 8.8 1525 8 10.6 1804 9 6.4 1452 10 13.8 1598 11 11.6 1743 12 10.2 1593 13 10.2 1412 14 8.3 1407 15 9.4 1481 16 9.0 1409 17 9.4 1644 18 8.5 1577 19 9.0 1586 20 9.7 1635 21 8.5 1512 22 8.5 1772 23 11.1 1259 24 9.7 1587 25 11.7 1545 N P MG eg g 1• 1 .15 0.193 0.140 1 .03 0.198 0.149 1 .01 0.184 0.141 1 . 12 0.157 0.150 1 .12 0.163 0.130 I .19 0.167 0.116 1 .10 0.184 0.147 1 .19 0.207 0 .143 0 .96 0.169 0.149 1 .20 0.197 0 .145 1 .16 0.198 0.152 1 .18 0.159 0.154 1 .12 0.176 0.148 0 .96 0.169 0.146 1 .04 0.160 0.167 I .16 0.164 0.154 1 .28 0.166 0.135 1 .06 0.165 0.143 1 .14 0.172 0.151 1 .25 0 .168 0.137 0 .99 0.161 0.137 1 .11 0.178 0 .170 1 .22 0.208 0.143 1 .16 0.157 0.163 1 .17 0.183 0.159 Appendix L (continued) STANDARD DEVIATION TRT ZN HN Ug g - x 1 5.8 511 2 1.8 432 3 2.7 447 4 4.2 918 5 2.5 780 6 3.8 1237 7 3.2 747 8 4.2 689 9 2.3 428 10 8.9 412 11 6.0 471 12 3.5 365 13 2.6 454 14 2.6 263 15 3.0 352 16 3.1 542 17 3.5 544 18 3.0 332 19 3.1 270 20 ' 3.5 375 21 3.9 281 22 2.0 343 23 3.5 305 24 2.7 363 25 4.6 415 N P HG eg g 0 .27 0.040 0 .030 0 .20 0 .045 0 .031 0 .20 0.055 0.033 0 .17 0.025 0 .021 0 .19 0.035 0.022 0 .21 0.034 0.023 0 .31 0.064 0.025 0 .27 0.043 0.015 0 .22 0.034 0.021 0 .25 0.026 0 .029 0 .21 0.042 0.024 0 .29 0 .047 0.012 0 .12 0.026 0.027 0 .20 0 .038 0.027 0 .27 0.051 0.031 0 .12 0.029 0 .033 0 .25 0.029 0.028 0 .27 0 .024 0 .021 0 .22 0.049 0.034 0 .22 0.048 0.031 0 .22 0.039 0.022 0 .20 0 .035 0.026 0 .15 0.024 0.029 0 .27 0 .031 0 .022 0 .21 0.041 0.040 248 Appendix L (continued) NUMBER OF CASES TRT ZN MN N P MG 1 9 9 9 9 9 2 9 9 9 9 9 3 9 9 9 9 9 4 10 10 9 10 10 5 9 9 9 9 9 6 9 9 9 9 9 7 10 10 10 10 10 8 7 7 7 7 7 9 9 9 9 9 9 10 10 10 10 10 10 11 9 9 9 9 9 12 8 8 8 8 8 13 9 9 9 9 9 14 8 8 8 8 8 15 8 8 8 8 8 16 7 7 7 7 7 17 10 10 10 10 10 18 8 8 8 8 8 19 9 9 9 9 9 20 10 10 10 10 10 21 9 9 9 9 9 22 9 9 9 9 9 23 9 9 9 9 9 24 10 10 10 10 10 25 9 9 9 9 9 Appendix L (continued) MEAN FE CU FLWTBR BI3 HTI3 HTI2 ug g - i 6 50.4 3.4 0.523 1.42 1.86 1.31 46.4 3.1 0.40 7 I .15 1.59 1 .15 52.0 3.2 0.364 1.14 1.59 1 .40 47.5 3.4 0.449 1.91 3 .14 1.21 50.4 3.3 0.506 1.77 2.65 1.32 55.9 3.6 0.646 1 .33 2.27 1.20 46.4 3.4 0.431 1.43 1.76 1.18 50.5 3.5 0.592 1.80 1.98 1.04 41.7 3.1 0.199 1.24 1.82 0.87 49.3 3.3 0 .665 1 .62 2.57 1.11 48.0 3.8 0.45 7 1.62 2.26 1.24 46.4 3.7 0 .428 1 .35 1.98 1.26 42.8 3.8 0.421 1.86 3.30 0.98 43 .3 3.4 0.371 1.75 2 .39 0.78 37.9 3.6 0.368 1.43 1.85 0.99 47.4 3.8 0.505 1.51 I .66 1 .04 54.3 3.6 0.540 1.66 1.75 1.19 46 .0 3.6 0.327 1.14 2 .35 1.01 41.7 3.9 0.459 1.46 2.11 0.98 43 .6 4.0 0 .515 1.30 1.60 1.25 44.8 3.3 0.410 1.46 2.00 0.83 46.8 3.7 0 .321 2.27 1.95 1.14 51.2 3.8 0.635 1.87 2.60 1.45 47.1 3.6 0.344 1.51 3.17 1.19 48.8 3.6 0.280 1.77 1.93 1.31 Appendix L (continued) STANDARD DEVIATION FE CU FLWTBR BI3 HTI3 HTI2 — P S g-l S 10.9 1.2 0.318 0.33 0.79 0.61 9.1 i . l 0.285 0 .36 0.50 0.60 12.5 0.9 0.195 0.51 1.01 0.89 18 .4 0.7 0 .219 0.65 2.29 0.77 20.2 0.7 0.358 0.59 1.55 0.77 9.8 0.8 0.459 0.50 1.57 0.56 12.0 1.1 0 .295 0.45 0.61 0 .40 10 .6 1.0 0.411 0.51 0 .65 0 .49 8.9 0.7 0.084 0.35 0.84 0.54 10.6 0.7 0.427 0.72 1.99 0.89 19.4 0.8 0.350 0.46 1.34 0.61 8.5 0.9 0.316 0.70 1.23 0.52 16.2 0.9 0.340 1.22 3.02 0.67 13 .3 0.9 0.226 0.70 3 .13 0.59 7.6 0.8 0.293 0.32 1.02 0.50 7.9 0.6 0.288 0.62 1.00 0.27 12.7 1.0 0.266 0.37 0.63 0.48 12.7 1.1 0.174 0 .39 1.66 0.31 8.4 0.7 0.277 0.20 1.25 0.48 6.3 0.9 0 .335 0.34 0.41 0.59 12.1 0.7 0.329 0.40 1.00 0.42 12.3 0.9 0.166 2.37 1.04 0.83 10.6 0.6 0.346 1.25 2.50 0.67 12 .7 0.9 0.233 0 .30 3 .18 0.80 15 .4 0.6 0.145 0.59 0.57 0.62 Appendix L (continued) NUMBER OF CASES FE CU FLWTBR BI3 HTI3 HTI2 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 10 9 10 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 10 10 10 7 7 7 7 7 7 9 9 9 9 9 9 10 10 10 10 10 10 9 9 9 9 9 9 8 8 8 8 8 8 9 9 9 9 9 9 8 8 8 8 7 8 8 8 8 8 8 8 7 7 7 7 7 7 10 10 10 10 10 10 8 8 8 8 8 8 9 9 9 9 9 9 10 10 10 10 10 10 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 10 10 10 9 9 9 9 8 9 Appendix L (continued) 5-86-86-86 MEAN TRT ZN MN ug g " 1 1 10.7 1262 2 11.6 1273 3 14.3 1289 4 11.9 1832 5 12.1 2172 6 15 .7 2933 7 10.1 1178 8 12.0 1525 9 8.8 1327 10 8.3 1083 11 10.5 1268 12 10.1 1240 13 79.3 1217 14 100.9 1066 15 132.3 867 16 14.1 1251 17 10.1 1425 18 11.2 1140 19 10.1 1272 20 10 .5 1094 21 11.2 1008 22 10.1 1270 23 15.5 1064 24 8.6 1091 25 11.4 1308 N P MG — -eg g 1 .02 0.155 0 .123 1 .02 0.193 0 .140 1 .12 0.183 0 .137 1 .21 0.153 0 .105 1 .14 0.176 0 .100 I .35 0.164 0 .124 1 .06 0.158 0 .125 1 .10 0.204 0 .134 0 .98 0.167 0 .150 0 .92 0.178 0 .140 1 .05 0.195 0 .140 0 .99 0.213 0 . 143 1 .26 0.188 0 .140 1 .13 0.195 0 .108 1 .04 0.188 0 .138 1 .08 0.182 0 . 106 1 .09 0.207 0 .137 I .21 0.192 0 .118 1 .04 0.182 0 .116 1 .02 0.178 0 .133 1 .16 0.176 0 .124 1 .01 0.150 0 .165 1 .67 0.214 0 .136 1 .02 0.138 0 .134 1 .11 0.182 0 .140 Appendix L (continued) STANDARD DEVIATION TRT ZN HN PS s - 1 1 5.8 137 2 4.9 209 3 7.1 548 4 2.3 510 5 5.2 794 6 5.8 1021 7 2.7 416 8 2.9 670 9 1.6 536 10 1.5 363 11 2.7 408 12 1.4 526 13 30.1 235 14 75.8 319 15 30.5 487 16 4.4 509 17 3.7 535 18 4.8 249 19 3.3 751 20 1.9 261 21 3.3 174 22 2.1 249 23 4.2 370 24 1.9 672 25 1.9 531 253 N P NG eg g 0.27 0.042 0.013 0.17 0.017 0 .050 0.26 0.025 0.012 0.08 0.041 0.019 0.24 0.036 0.012 0. 12 0 .025 0.040 0.34 0.032 0.019 0 .14 0.073 0.026 0.05 0.055 0.044 0.12 0.056 0.026 0.19 0.048 0.034 0.22 0.049 0.033 0.16 0.028 0.018 0.27 0.031 0 .022 0.24 0.043 0.019 0.14 0.047 0.011 0.15 0.055 0.023 0.24 0 .054 0.022 0.13 0.015 0.015 0.06 0.043 0.026 0.19 0.017 0.026 0.17 0.024 0.040 0.22 0.066 0.052 0.15 0.030 0.038 0.11 0.052 0.016 254 Appendix L (continued) NUMBER OF CASES TRT ZN MN N P MG 1 4 4 4 4 4 2 4 4 4 4 4 3 3 3 3 3 3 4 4 4 4 4 4 5 5 5 5 5 5 6 5 5 5 5 5 7 4 4 4 4 4 8 5 5 5 5 5 9 3 3 3 3 3 10 4 4 4 4 4 11 4 4 4 4 4 12 4 4 4 4 4 13 4 4 4 4 4 14 4 4 4 4 4 15 5 5 5 5 5 16 5 5 5 5 5 17 3 3 3 3 3 18 5 5 5 5 5 19 5 5 5 5 5 20 4 4 4 4 4 21 5 5 5 5 5 22 4 4 4 4 4 23 5 5 5 5 5 24 5 5 5 5 5 25 5 5 5 5 5 Appendix L (continued) MEAN FE CU B AFE FLWTBE BI2 S PS 6 40.2 2.9 22.3 26.7 0.169 1.01 4 i .9 2.9 22 .1 27.8 0.201 1 .03 40.5 3.3 23.1 25 .9 0.171 0.87 45 .5 3.6 20.1 30 .2 0 .302 1.25 40.0 3.1 24.2 29.2 0.250 1.21 40.0 3.6 23 .9 28.2 0.367 1.23 32.1 3.5 35.7 28.5 0.256 1.08 32.1 3.0 38 .5 25.8 0.175 0.99 44.0 2.7 47.1 27.7 0.189 0.86 32.1 3.0 40.9 24.2 0.193 1.09 36.6 2.9 51.9 25.6 0.168 0.98 36 .6 3.1 52.6 27.3 0.250 1.08 46.4 4.9 22.1 30.0 0.184 0.97 36 .6 3.0 22.4 30 .3 0.173 0.87 40.7 2.8 26.8 32.6 0.171 0.83 39.3 3.1 21.1 31.3 0.277 1.10 45 .2 3.8 24.5 33.9 0.160 0.95 36 .4 3.8 23.3 32.1 0.291 0 .98 32.1 3.5 24.9 28.9 0.198 0.93 32.1 2.9 25 .5 29. 7 0 .174 0 .94 32.1 3.4 24.8 .26.4 0 .200 0.98 30 .3 3.2 23.5 29.6 0.181 0.93 46.4 4.0 45 .8 34.8 0.394 1.58 51.4 3.6 23.2 32.7 0 .146 0.88 39.3 3.1 28.4 26.2 0.198 0.96 256 Appendix L (continued) STANDARD DEVIATION FE CU B AFE FLWTBR BI2 U g g 1- 8 12.2 0.7 1.3 7.4 0.045 0.26 6.1 0.6 7.6 6.2 0.081 0 .38 8.2 0.2 3.5 3.0 0.028 0.32 11.8 0.7 5.2 3.2 0.096 0.35 8.9 0.7 4.8 7.7 0.134 0.52 6.4 0.7 5.3 4.2 0.187 0 .39 13.0 1.6 12.2 3.1 0.107 0.36 7.1 0.6 17.8 6.3 0.078 0.31 7.4 0.5 11.3 4.1 0.021 0.21 14.9 0.4 13.3 9.0 0.075 0.35 3.4 0.3 10.1 5.0 0.037 0.37 5.4 0.3 15 .2 4.4 0.210 0.39 15 .4 1.3 3.0 5.9 0.078 0.22 8.4 l . l 2.3 4.6 0 .052 0.31 15.5 1.3 4.5 10.9 0.093 0.37 11.6 0.4 1.7 5.5 0.149 0.36 10.3 0.5 4.2 9.0 0.045 0.27 8.5 1.0 6.3 6.8 0.072 0.35 12 .6 0.5 5.7 7.6 0.071 0.26 12.7 0.7 9.2 2.4 0.052 0.35 7.1 0.6 5.5 7.3 0.100 0.35 7.4 0.4 1.9 7.0 0 .049 0.23 9.1 0.6 10.9 6.4 0.162 0.67 12.5 0.6 2.4 3.5 0.048 0.21 6.7 0.6 6.0 5.3 0.054 0.27 \ 257 Appendix L (continued) NUMBER OF CASES FE CU B AFE FLWTBR BI2 4 4 4 4 4 24 4 4 4 4 4 27 3 3 3 3 3 27 4 4 4 4 4 27 5 5 5 5 5 25 5 5 5 5 5 27 4 4 4 4 4 24 5 5 5 5 5 30 3 3 3 3 3 23 4 4 4 4 4 24 4 4 4 4 4 27 4 4 4 4 4 24 4 4 4 4 4 24 4 4 4 4 4 24 5 5 5 5 5 26 5 5 5 5 5 30 3 3 3 3 3 21 5 5 5 5 5 27 5 5 5 5 5 21 4 4 4 4 4 29 5 5 5 5 5 30 4 4 4 4 4 24 5 5 5 5 5 24 5 5 5 5 5 30 5 5 5 5 5 27 Appendix L (continued) 5-86-87-87 MEAN TRT ZN MN ug g " 1 1 11.9 1190 2 12.6 1296 3 15 .2 1342 4 11.2 2487 5 12.6 2817 6 12.5 3518 7 11.3 1603 8 11.5 1322 9 8.8 1439 10 9.9 1359 11 12.0 1626 12 11.6 1301 13 11.9 1581 14 15 .4 1540 15 16.5 1533 16 11.5 1515 17 9.9 1285 18 10.5 1231 19 12 .5 1463 20 12.4 112 7 21 12.2 1551 22 12 .0 1434 23 14.7 962 24 9.9 1142 25 11.8 1243 258 N P MG -eg g *•• 1 .04 0.163 0 .139 1 .12 0.185 0 .139 1 .04 0.173 0 .148 1 .16 0.157 0 .114 1 .12 0.171 0 .113 1 . 16 0.159 0 .117 1 .20 0.164 0 .133 I .15 0.177 0 .147 1 .03 0.143 0 .140 1 .09 0.159 0 .159 1 .23 0.175 0 .139 1 .12 0.181 0 .160 1 .12 0.165 0 .147 1 .19 0.174 0 .124 1 .22 0.215 0 .154 1 . 10 0.173 0 .133 1 .11 0.162 0 .139 1 .17 0.192 0 .139 1 .18 0.170 0 .130 1 .16 0.173 0 .143 1 .21 0.179 0 .145 1 .20 0 .172 0 . 146 1 .32 0.242 0 .149 1 .07 0.148 0 .141 1 .15 0.166 0 .131 Appendix L (continued) STANDARD DEVIATION TRT ZN MN ug g-X 1 5.2 282 2 4.7 217 3 5.7 573 4 3.1 592 5 4.2 651 6 4.4 881 7 3.5 574 8 2.8 618 9 1.8 233 10 2.5 523 11 3.5 632 12 3.2 45 4 13 2.9 683 14 4.9 550 15 4.5 656 16 2.6 344 17 3.9 382 18 4.6 568 19 5.1 577 20 3.5 231 21 2.7 462 22 2.9 418 23 2.3 383 24 3.6 571 25 3.6 666 259 N P MG cg g 0 .28 0.037 0.026 0 .25 0.028 0.036 0 .23 0.044 0.021 0 .16 0.029 0.015 0 .20 0.038 0.018 0 .12 0.035 0.027 0 .26 0.030 0.037 0 .18 0.066 0 .026 0 .16 0.037 0.016 0 .23 0.051 0 .031 0 .28 0.053 0.028 0 .22 0.047 0.025 0 .17 0.041 0.026 0 .24 0.059 0.018 0 .19 0.049 0.029 0 .19 0.047 0.031 0 .19 0.053 0.026 0 .28 0.043 0.026 0 .22 0.027 0.024 0 .24 0.054 0.022 0 .12 0.037 0.027 0 .27 0.048 0.040 0 .15 0.037 0.040 0 .22 0.032 0.028 0 .17 0.062 0.024 260 Appendix L (continued) NUMBER OF CASES TRT ZN MN N P MG 1 10 10 10 10 10 2 8 8 8 8 8 3 9 9 9 9 9 4 10 10 10 10 10 5 10 10 10 10 10 6 10 10 10 10 10 7 10 10 10 10 10 8 10 10 10 10 10 9 10 10 10 10 10 10 9 9 9 9 9 11 10 10 10 10 10 12 10 10 10 10 10 13 10 10 10 10 10 14 9 10 10 10 10 15 10 10 10 10 10 16 10 10 10 10 10 17 10 10 10 10 10 18 10 10 10 10 10 19 10 10 10 10 10 20 9 9 9 9 9 21 10 10 10 10 10 22 10 10 10 10 10 23 9 9 9 9 9 24 10 10 10 10 10 25 10 10 10 10 10 Appendix L (continued) MEAN FE CU FLWTBR BI3 HTI3 HTI2 Ug g - 1  S 42.1 3.1 0.268 1.30 1.76 1.08 44.6 3.4 0.393 1 .40 1 . 78 1.38 41.7 3.2 0.258 1.15 1.63 1.17 43 .2 3.5 0.317 1.22 1.74 1.82 40.7 3.4 0.340 1.17 2.03 1.52 42 .5 3.6 0.316 1.11 1.50 1.74 38.9 3.6 0.366 1.35 1.31 1.58 38.9 3.6 0.257 1.59 2.09 1.35 37.1 3.1 0.272 1.23 1.64 1.12 38.9 3.3 0.291 1.14 1.63 1 .24 44.6 3.7 0.326 1.34 1.43 1.69 41.4 3.2 0.314 1.42 1.46 1.27 38.6 3.4 0.306 1.91 2.36 1.02 42.8 3.4 0 .292 1.60 2.37 0.98 43.2 3.5 0.314 1.77 2.68 0.64 41.8 3.0 0.283 1.60 1.73 1. 70 38.9 3.3 0.223 1.37 2.22 0.78 42 .5 3.5 0.278 1.20 1.43 1.09 45 .0 3.5 0.301 1.38 1.75 1.16 40.9 3.3 0.308 1 .29 2 .06 1 .03 44.3 3.5 0.272 1.41 1.94 1.28 42 .1 3.2 0.307 1.25 1.26 1.03 53.7 3.6 0.742 1.94 1.82 1.49 38.9 3.0 0.233 1.53 2.06 1 .08 52.7 3.9 0.288 1.39 1.69 1.25 Appendix L (continued) STANDARD DEVIATION FE CU FLWTBR BI3 HTI3 HTI2 PS g-X S 7.3 0.7 0.181 0.47 0.88 0.58 8.3 0.8 0.222 0.44 1.04 0.91 3.6 0.4 0.121 0.42 1.41 0.98 7.0 0.6 0.089 0.30 0.44 0.76 20.6 0.9 0.157 0.38 1.26 0.71 12.0 0.8 0.103 0 .22 0.41 0.53 9.3 1.0 0.217 0.36 0.46 0.89 9.4 1.2 0.136 0.95 1.33 0.64 7.7 0.9 0.108 0.35 0.69 0.43 5.5 0.9 0.156 0 .42 0.59 0.46 6.8 1.3 0.152 0.20 0.28 0.87 7.9 1.2 0 .180 0.69 0.86 0.57 5.0 1.1 0.152 0.89 1.28 0.56 9.7 0.5 0.104 0.54 1.20 0.26 8.3 0.8 0.170 0.43 1.56 0.30 11.7 0.8 0.126 1.14 0.48 0.94 11.8 0.9 0.083 0.45 1.49 0.32 8.5 1.3 0.110 0.29 0.46 0.46 8.4 1.0 0.151 0.41 0.56 0.67 6.0 0.6 0.165 0.54 1.09 0.41 10.3 0.8 0.081 0.42 0.98 0.47 6.0 0.7 0.193 0.27 0.67 0.36 7.0 0.7 0.306 1.11 0.83 0.48 7.8 0.7 0.116 0.79 1.14 0.43 12.0 0.9 0.140 0.14 0.56 0.61 263 Appendix L (concluded) NUMBER OF CASES FE CU FLWTBR BI3 HTI3 HTI2 10 10 10 10 to to 8 8 8 8 8 8 9 9 9 9 9 9 10 10 10 10 10 10 10 10 10 to to 10 10 10 10 10 10 10 10 10 to 10 10 10 10 10 10 10 10 10 10 10 to 10 10 10 9 9 9 9 9 9 10 10 to 10 10 10 10 10 10 10 10 10 10 10 10 to to 10 10 10 10 10 10 10 10 10 to to to 10 10 10 10 to 10 10 10 10 to to to 10 10 10 10 10 10 10 to 10 10 10 to 10 9 9 9 9 9 9 10 10 10 10 to to 10 10 10 10 10 10 9 9 9 9 9 9 10 10 10 10 10 10 10 10 10 to 10 to Symbols SOS = s u l p h a t e - s u l p h u r TOTS = t o t a l sulphur FLWTBR = f o l i a r mass (g) per shoot of the c u r r e n t year's growth Bl = shoot increment r a t i o HTI = height increment r a t i o 1 = growth increment i n 1985/growth increment i n 1984 2 = growth increment i n 1986/growth increment i n 1985 3 = growth increment i n 1987/growth increment i n 1986 264 APPENDIX M. FOLIAR NUTRIENT DATA FOR ZINC AND AND AGE. Appendix Ml. S i t e 1. MEAN TRT ZN1 MN1 ZN2 3 11.0 2971 24.4 4 13.6 1352 48.7 5 15.4 1143 135.5 7 11.5 1231 19.8 8 11.0 1228 17.9 11 8.8 992 13.0 12 9.7 1139 18.5 STANDARD DEVIATION TRT ZN1 MN1 ZN2 3 4.6 1278 5.7 4 2.3 650 20.2 5 5.7 291 40.7 7 3.5 352 6.6 8 3.7 460 4.5 11 2.3 200 2.0 12 3.2 395 11.8 STANDARD ERROR TRT ZN1 MN1 ZN2 3 1.6 452 2.1 4 1.0 265 7.6 5 2.2 110 15.4 7 1.2 124 2.5 8 1.3 163 1.6 11 0.8 67 0.7 12 l . l 132 3.7 NUMBER OF CASES TRT ZN1 MN1 ZN2 3 8 8 7 4 6 6 7 5 7 7 7 7 8 8 7 8 8 8 8 11 9 9 9 12 9 9 10 MANGANESE (ug g-1) WITH TIME ZN3 MN2 MN3 10.4 4610 3059 51.2 1481 1146 .44.4 1851 1200 5.1 1733 1195 2.5 1464 1203 2.1 1382 1067 2.2 1432 1019 ZN3 MN2 MN3 4.9 2001 1320 17.0 850 289 47.0 722 333 3.1 474 331 1.1 630 340 0.8 395 334 1.4 594 311 ZN3 MN2 MN3 1.8 756 46 7 6.9 321 118 L7.8 273 126 1.2 179 117 0.4 223 120 0.3 132 111 0.5 188 98 ZN3 MN2 MN3 7 7 8 6 7 6 7 7 7 7 7 8 7 8 8 6 9 9 8 10 10 265 Appendix M2. S i t e 2. MEAN TRT ZN1 MN1 ZN2 ZN3 MN2 MN3 3 12.4 3875 15 .5 3.2 5920 3196 4 11.5 1527 35 .2 53.0 1704 1676 5 19.6 1141 78.7 139.6 1687 1251 7 8.8 1728 15.6 2.0 1082 1633 8 11.4 1181 17.0 2.5 1617 1037 11 10.1 1249 9.5 2.0 1726 1048 12 11.1 1932 14.8 3.0 2365 1474 STANDARD DEVIATION TRT ZN1 MN1 ZN2 ZN3 MN2 MN3 3 5.6 1429 5.7 3.0 2804 1495 4 5.6 1031 18.8 21.9 1322 558 5 8.8 434 34.2 746 101 7 3.4 1025 3.0 0.0 1030 482 8 2.7 601 7.0 2.1 737 677 11 4.2 357 3.5 1.7 206 175 12 4.8 589 8.2 2.2 956 666 STANDARD ERROR TRT ZN1 MN1 ZN2 ZN3 MN2 MN3 3 1.9 476 2.0 1.3 991 498 4 2.1 390 8.4 9.8 591 249 5 3.9 194 15 .3 334 71 7 1.7 513 2.1 0.0 728 278 8 1.1 245 3.1 1.5 330 276 11 2.1 179 2.0 1.0 119 87 12 1.7 208 2.9 1.1 338 222 NUMBER OF CASES TRT ZN1 MN1 ZN2 ZN3 MN2 MN3 3 9 9 8 5 8 9 4 7 7 5 5 5 5 5 5 5 5 1 5 2 7 4 4 2 2 2 3 8 6 6 5 2 5 6 11 4 4 3 3 3 4 12 8 8 8 4 8 9 1 = one-year o l d f o l i a g e formed and c o l l e c t e d i n 1986, two years a f t e r treatment. 2 = two-year-old f o l i a g e formed i n 1985 and c o l l e c t e d i n 1986, two years a f t e r treatment. 3 = one-year-old f o l i a g e formed and c o l l e c t e d i n 1985, one year a f t e r treatment. Equations from Appendix D were used to convert the o r i g i n a l AA values to t h e i r e q u i v a l e n t values f o r the ICP which are presented here. 266 A P P E N D I X N . S C A T T E R P L O T S OF HEIGHT INCREMENT V E R S U S F O L I A R Z I N C . A p p e n d i x N . l . S c a t t e r p l o t o f f i r s t y e a r t o t a l h e i g h t i n c r e m e n t (cm) ( i n 1 9 8 6 ) v e r s u s f i r s t y e a r f o l i a r z i n c l e v e l s ( u g g _ t ) ( f o r c a s e s w h e r e Zn <100 pg g - 1 ) on s i t e 5 . 267 A p p e n d i x N . 2 . S c a t t e r p l o t o f s e c o n d y e a r t o t a l h e i g h t i n c r e m e n t (cm) ( i n 1 9 8 7 ) v e r s u s s e c o n d y e a r f o l i a r z i n c l e v e l s ( u g g - M on s i t e 5 . 268 APPENDIX 0 . SCATTER PLOTS OF FOLIAR NITROGEN VERSUS FOLIAR ZINC. 1.80 ~z 1.60 z LO = j . „ n 1 c r a H e r u l o t of f o l i a r n i t r o g e n (cg g" 1) versus tn"r"i2; l; M S""".«'c.:.. where Z . 130 ps f . r c - n t ye a r ' s f o l i a g e ( i n 1985) from s i t e 2. 269 1.6 - i CD 1.4 i CO CD C 1.2 i CD CD O 1.0 H O "5 0.8 y = 0.8 + 0.038x - 0.0009x R = 0.21 0.6 i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i II i i i i i i | 0.0 10.0 20.0 30.0 40.0 Fol iar Z inc 1 9 8 6 A p p e n d i x 0 . 2 . S c a t t e r p l o t o f f o l i a r n i t r o g e n ( e g g _ 1 ) v e r s u s f o l i a r z i n c ( pg g - M u s i n g a l l t r e a t m e n t s ( f o r c a s e s where Zn <30 pg g - 1 - ) f o r c u r r e n t y e a r ' s f o l i a g e ( i n 1986 ) f r o m s i t e 2 . 1.8 -2 A p p e n d i x 0 . 3 . S c a t t e r p l o t o f f o l i a r n i t r o g e n ( c g g _ 1 ) v e r s u s f o l i a r z i n c ( p g g - 1 - ) u s i n g a l l t r e a t m e n t s f o r c u r r e n t y e a r ' s f o l i a g e ( i n 1 9 8 6 ) f r o m s i t e 3 . 271 2.0 n CD 1.6 -3 OO C 1.2 i CD cn O 0.8 H ~0 0.4 ^ l_L_ 0.0 y = 0.137x - 0.0036Lx R = 0.46 I | | | I I I I I I I I I I I I I I I I I I I I I M I | I I I I M I I I | I I I I I I I I I | M I I I I I I I | 0 0 5 0 10.0 15.0 20.0 25.0 30.0 Fol iar Z inc 1 9 8 6 A p p e n d i x 0.4. S c a t t e r p l o t o f f o l i a r n i t r o g e n ( c g g _ t ) v e r s u s f o l i a r z i n c ( p g g _ t ) ( f o r c a s e s w h e r e Zn <30 pg g - 1 ) f o r c u r r e n t y e a r ' s f o l i a g e ( i n 1 9 8 6 ) f r o m s i t e 4. 272 A p p e n d i x 0 . 5 . S c a t t e r p l o t o f f o l i a r n i t r o g e n ( c g g - 1 ) v e r s u s f o l i a r z i n c ( p g g - 1 ) u s i n g a l l t r e a t m e n t s o f c u r r e n t y e a r ' s f o l i a g e ( i n 1 9 8 7 ) f r o m s i t e 4 . A p p e n d i x 0 . 6 . S c a t t e r p l o t o f f o l i a r n i t r o g e n ( e g g - 1 ) f o l i a r z i n c ( u g g - M ( f o r c a s e s w h e r e Zn <40 pg g _ 1 ) o f y e a r ' s f o l i a g e ( i n 1986 ) f r o m s i t e 5 . 274 A p p e n d i x 0 . 7 . S c a t t e r p l o t o f f o l i a r n i t r o g e n ( c g g _ 1 ) v e r s u s f o l i a r z i n c (pg g - M u s i n g a l l t r e a t m e n t s o f c u r r e n t y e a r ' s f o l i a g e ( i n 1 9 8 7 ) f r o m s i t e 5 .

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