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Some aspects of boron, copper and iron nutrition of lodgepole pine and Douglas-fir Majid, Nik Muhamad 1984

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SOME ASPECTS OF BORON, COPPER AND IRON NUTRITION OF LODGEPOLE PINE AND DOUGLAS-FIR by NIK MUHAMAD MAJID (Dip. A g r i c , Malaya, 1971) (B.S., Louisiana State University, 1973) (M.F., Louisiana State University, 1975) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Soil Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1984 © Nik Muhamad Majid, 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f S o i l S c i e n c e  The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main M a l l V a n c o u v e r , C a n a d a V6T 1Y3 D a t e J u l y 3 , 1 9 8 4 i i ABSTRACT This thesis reports the findings of two complementary i n v e s t i g a -tions on boron, copper and i r o n n u t r i t i o n of lodgepole pine and Douglas-f i r . The primary aim of the f i r s t study was to determine the c r i t i c a l l e v e l s of boron, copper, t o t a l i r o n and "active" i r o n f o r lodgepole pine grown under co n t r o l l e d conditions i n the greenhouse. The second study, conducted at f i v e d i f f e r e n t f i e l d locations i n i n t e r i o r B r i t i s h Columbia, involved f o l i a r a p p l i c a t i o n of copper sulphate, ferrous sulphate and urea to lodgepole pine; and ferrous sulphate and urea to Douglas-fir. The main objective of the f i e l d study was to assess tree growth and f o l i a r nutrient responses to f o l i a r - a p p l i e d n u t r i e n t s . Both studies involved the a p p l i c a t i o n of nitrogen to investigate i t s s i g n i f i c a n c e f o r the micronutrients. The findings from the greenhouse experiments indicated that f or lodgepole pine, the c r i t i c a l range, expressed as concentration i s 7 to 16 ppm for boron and 2 to 3 ppm for copper. The lower value i n each case i s the c r i t i c a l value. For "active" and t o t a l i r o n , the c r i t i c a l values are 32 and 44 ppm, r e s p e c t i v e l y . Concentrations at or below the c r i t i c a l value, i n each case, may be associated with acute deficiency of the nutrient element. Concentrations above the c r i t i c a l value imply that the nutrient i s at a l e v e l s u f f i c i e n t f o r optimum or near-optimum growth. Results from the f i e l d experiments with lodgepole pine indicated f a i r l y comparable values to those from the greenhouse. The c r i t i c a l value for copper was found to be 4 ppm f o r copper and 29 ppm f o r active i i i i r o n . It was also suggested that copper t o x i c i t y i n lodgepole pine might occur whenever f o l i a r copper concentration exceeds 17 ppm. F o l i a r nutrient a p p l i c a t i o n proved to be a quick and e f f e c t i v e means of r a i s i n g copper and i r o n concentrations to adequate l e v e l s i n lodgepole pine f o l i a g e ; and i r o n i n Douglas-fir f o l i a g e where these nutrients were d e f i c i e n t . F o l i a r a p p l i c a t i o n of urea was e f f e c t i v e i n r a i s i n g f o l i a r nitrogen concentration i n lodgepole pine, but not i n Douglas-fir. Combined nutrient applications were more e f f e c t i v e than i n d i v i d u a l nutrient a p p l i c a t i o n . However, these e f f e c t s on f o l i a r nutrients were only temporary and did not l a s t beyond the year of a p p l i -cation, except i n Douglas-fir which seemed able to r e t a i n more applied i r o n i n the f o l i a g e than did lodgepole pine, during the second year of growth• Shoot growth and blomass production i n both species responded greatly to treatments only during the second growing season following f e r t i l i z a t i o n . Lodgepole pine needle length at one of the s i t e s also showed a s i g n i f i c a n t p o s i t i v e response to treatments i n the second year of growth. The highest response was obtained from treatments that caused minimal f o l i a r scorching. No s i g n i f i c a n t p o s i t i v e tree growth response was detected during the year of f e r t i l i z e r a p p l i c a t i o n . The safe a p p l i c a t i o n dosage (where no f o l i a r i n j u r y was evident) of copper sulphate and ferrous sulphate to lodgepole pine was 0.1 and 2 percent, r e s p e c t i v e l y . No f o l i a r i n j u r y was observed with 4 percent ferrous sulphate applied to Douglas-fir. Nitrogen applied at 2 percent urea did not cause any f o l i a r burn i n e i t h e r species. The a p p l i c a t i o n of 1 percent copper sulphate was extremely to x i c to lodgepole pine; 4 percent ferrous sulphate caused moderate f o l i a r burn. i v Nitrogen absorbed by the root system (greenhouse experiment) appeared to have an antagonistic e f f e c t on f o l i a r boron, copper, t o t a l and active i r o n i n lodgepole pine. The concentration and content of these micronutrients i n the f o l i a g e decreased as f o l i a r nitrogen increased as a r e s u l t of increasing the nitrogen supply. F o l i a r feeding of urea ( f i e l d experiment), on the other hand, did not seem to have any p h y s i o l o g i c a l i n t e r a c t i o n with f o l i a r copper. In f a c t , there was a sy n e r g i s t i c e f f e c t of urea on f o l i a r i r o n . The l e v e l of t o t a l and active forms of i r o n i n the f o l i a g e was increased as a r e s u l t of urea a p p l i c a t i o n . TABLE OF CONTENTS v Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES xi LIST OF APPENDICES xiv ACKNOWLEDGEMENTS xvi CHAPTER 1. GENERAL INTRODUCTION 1 CHAPTER 2. LITERATURE REVIEW 3 A. Nutrient Requirement Evaluations 3 1. F e r t i l i z e r Field Trials 3 2. F e r t i l i z e r Pot Trials 4 3. Visual Symptoms 4 4. Soil Analysis 5 5. Foliar Analysis 6 B. Expression of Foliar Nutrient Composition 9 C. Nutrient Requirements of Conifers 12 CHAPTER 3. GREENHOUSE EXPERIMENTS 15 A. Introduction 15 B. Methods and Materials 15 1. Experimental Design 15 2. Pot Arrangement 16 3. Greenhouse Environment 17 4. Preparation and Maintenance of Growth Medium 17 v i Page 5. Germination and Thinning 19 6. Composition of Nutrient Solutions 20 7. Nutrient Application 21 8. Harvesting, Sample Measurement and Preparation 24 9. Chemical Analysis 24 10. Stat i s t i c a l Analysis 26 C. Results and Discussion 26 1. Boron Experiment 26 (a) Visual Symptoms 26 (b) C r i t i c a l Level 33 2. Copper Experiment 33 (a) Visual Symptoms 33 (b) C r i t i c a l Level 41 3. Iron Experiment 41 (a) Visual Symptoms 41 (b) C r i t i c a l Level 43 D. Summary of Greenhouse Experiments 51 CHAPTER 4. FIELD EXPERIMENT 52 A. Introduction 52 B. Methods and Materials 54 1. Site Descriptions 54 (a) Site Locations 54 (b) Stand Characteristics 56 (c) Soil Characteristics 57 2. Experimental Design 57 3. F e r t i l i z e r Application 60 v i i Page 4. F i e l d Sampling and Measurements 64 5. Sample Preparation and Measurements 65 6. Chemical Analysis of F o l i a r Samples 65 7. S o i l Sample Preparation and Analysis • 66 8. Assessment of F e r t i l i z e r Response 66 (a) Evaluation of Shoot Length and F o l i a r Mass Response 67 (b) Evaluation of F o l i a r Nutrient Response . 68 (c) F o l i a r Nutrient Status Interpretation .. 69 9. S t a t i s t i c a l Analysis 69 C. Results and Discussion 70 1. F o l i a r Scorching 71 (a) Copper Treatments 71 (b) Iron Treatments 72 (c) Nitrogen Treatments 73 (d) Causes of Needle Burn 73 2. Tree Growth Responses 75 (a) Shoot Growth 75 ( i ) Lodgepole Pine 75 ( i i ) Douglas-fir 81 (b) F o l i a r Mass 83 ( i ) Lodgepole Pine 83 ( i i ) Douglas-fir 85 3. F o l i a r Nutrient Responses 87 (a) Copper 87 (b) Active Iron 101 ( i ) Lodgepole Pine 101 ( i i ) Douglas-fir 107 v i i i Page (c) Nitrogen 109 (i) Lodgepole Pine 109 ( i i ) Douglas-fir 115 D. Summary of Field Experiment 116 CHAPTER 5. CONCLUSIONS 118 REFERENCES CITED 122 XX LIST OF TABLES Table Page 1 F o l i a r nutrient values useful i n i n d i c a t i n g nutrient status of conifers 13 2 Chemical analysis of the leachate from the sand used as the growth medium 18 3 Thinning frequency, dates and number of seedlings remaining a f t e r each thinning 20 4 Concentration of manipulated elements i n nutrient solutions 21 5 Concentration and source of elements i n standard nutrient s o l u t i o n f o r l e v e l 1 nitrogen supply (10 ppm) 22 6 Concentration and source of elements i n standard nutrient s o l u t i o n f o r l e v e l 2 nitrogen supply (100 ppm) 23 7 F o l i a r boron composition, mass of 100 needles and seedling height 31 8 Analysis of variance of f o l i a r nutrient composi-t i o n with varying supply l e v e l s of boron and nitrogen 32 9 F o l i a r copper composition, mass of 100 needles and seedling height 39 10 Analysis of variance of f o l i a r nutrient composi-t i o n with varying supply l e v e l s of copper and nitrogen 40 11 To t a l and active i r o n composition, mass of 100 needles and seedling height 47 12 Analysis of variance of f o l i a r nutrient composi-t i o n with varying supply l e v e l s of i r o n and nitrogen 48 13 Some chemical c h a r a c t e r i s t i c s of s o i l p r o f i l e s of the study s i t e s 58 14 Some physi c a l s o i l c h a r a c t e r i s t i c s of the study s i t e s 59 X Table Page 15 F e r t i l i z e r treatments f o r the main (1981) t r i a l .... 61 16 F e r t i l i z e r treatments for the repeat (1982) t r i a l .. 62 17 Chemical analysis of water used for preparing f e r t i l i z e r s o l u t i o n 63 18 Analysis of variance of tree growth response variables for lodgepole pine and Douglas-fir 76 19 Analsysis of variance f o r f o l i a r nutrient response of lodgepole pine (main t r i a l ) 88 20 Percentage variance components i n r e l a t i o n to f o l i a r nutrient responses for lodgepole pine (main t r i a l ) 89 21 Analysis of variance for f o l i a r nutrient responses of lodgepole pine (repeat t r i a l ) 90 22 Percentage variance components i n r e l a t i o n f o l i a r nutrient responses for lodgepole pine (repeat t r i a l ) 90 23 Analysis of variance for f o l i a r nutrient responses of Douglas-fir (main t r i a l ) 91 24 Percentage variance components i n r e l a t i o n f o l i a r nutrient responses f o r Douglas-fir (main t r i a l ) .... 91 25 Analysis of variance f o r f o l i a r nutrient responses of Douglas-fir (repeat t r i a l ) 92 26 Treatment and year e f f e c t s on f o l i a r nitrogen concentration (%) of Douglas-fir 115 x i LIST OF FIGURES Figure Page 1 Generalized r e l a t i o n s h i p between growth and tissue nutrient concentration 7 2 Lodgepole pine seedlings without boron supply 28 3 D i f f e r e n t i a l growth response of lodgepole pine seedlings to increasing boron supply at low nitrogen l e v e l 29 4 D i f f e r e n t i a l growth response of lodgepole pine seedlings to increasing boron supply at high nitrogen l e v e l 30 5 Relationship between f o l i a r boron composition and seedling growth (lodgepole pine) 34 6 Lodgepole pine seedlings without copper supply 36 7 D i f f e r e n t i a l growth response of lodgepole pine seedlings to increasing copper supply at low nitrogen l e v e l 37 8 D i f f e r e n t i a l growth response of lodgepole pine seedlings to increasing copper supply at high nitrogen l e v e l 38 9 Relationship between f o l i a r copper composition and seedling growth (lodgepole pine) 42 10 Lodgepole pine seedlings without i r o n supply 44 11 D i f f e r e n t i a l growth response of lodgepole pine seedlings to increasing i r o n supply at low nitrogen l e v e l 45 12 D i f f e r e n t i a l growth response of lodgepole pine seedlings to increasing i r o n supply at high nitrogen l e v e l 46 13 Relationship between f o l i a r t o t a l i r o n composition and seedling growth (lodgepole pine) 49 14 Relationship between f o l i a r a ctive i r o n composition and seedling growth (lodgepole pine) 50 15 Map showing l o c a t i o n of the study s i t e s 55 x i i Figure Page 16 Second-year shoot growth r a t i o i n r e l a t i o n to t r e a t -ments at S i t e 1 (lodgepole pine) 77 17 Second-year shoot growth r a t i o i n r e l a t i o n to t r e a t -ments at Site 2 (lodgepole pine) 78 18 Second-year shoot growth r a t i o i n r e l a t i o n to t r e a t -ments at Si t e 3 (lodgepole pine) 79 19 Second-year shoot growth r a t i o i n r e l a t i o n to t r e a t -ments at Site 4 (lodgepole pine) 80 20 Second-year shoot growth r a t i o i n r e l a t i o n to t r e a t -ments at S i t e 5 (Douglas-fir) 82 21 F o l i a r mass of lodgepole pine i n the second growing season i n r e l a t i o n to treatments 84 22 F o l i a r mass of Douglas-fir i n the second growing season i n r e l a t i o n to treatments 86 23 F o l i a r copper concentration i n r e l a t i o n to treatments at Site 2 (lodgepole pine) 93 24 F o l i a r copper concentration i n r e l a t i o n to treatments at S i t e 3 (lodgepole pine) 94 25 F o l i a r copper concentration i n r e l a t i o n to treatments at Site 4 (lodgepole pine) 95 26 F o l i a r copper concentration i n r e l a t i o n to treatments at S i t e 1 (lodgepole pine) 97 27 F o l i a r copper concentration i n r e l a t i o n to treatments at S i t e 5 (Douglas-fir) 98 28 F o l i a r a c t i v e i r o n concentration i n r e l a t i o n to t r e a t -ments at S i t e 1 (lodgepole pine) 102 29 F o l i a r a c t i v e i r o n concentration i n r e l a t i o n to t r e a t -ments at Si t e 2 (lodgepole pine) 103 30 F o l i a r a c t i v e i r o n concentration i n r e l a t i o n to t r e a t -ments at S i t e 3 (lodgepole pine) 104 31 F o l i a r a c t i v e i r o n concentration i n r e l a t i o n to t r e a t -ments at Site 4 (lodgepole pine) 105 x i i i Figure Page 32 F o l i a r active i r o n concentration i n r e l a t i o n to t r e a t -ments at Site 5 (Douglas-fir) 108 33 F o l i a r nitrogen concentration i n r e l a t i o n to t r e a t -ments at Site 1 (lodgepole pine) 110 34 F o l i a r nitrogen concentration i n r e l a t i o n to treatments at Site 2 (lodgepole pine) I l l 35 F o l i a r nitrogen concentration i n r e l a t i o n to treatments at Site 3 (lodgepole pine) 112 36 F o l i a r nitrogen concentration i n r e l a t i o n to treatments at Site 4 (lodgepole pine) 113 xiv LIST OF APPENDICES Appendix Page A . l Modified Parkinson and A l l e n Digestion for plant tis s u e analysis 132 A.2 N i t r i c a c i d digestion for analysis of copper and i r o n i n plant tissue 133 A. 3 Procedure for a c t i v e i r o n determination i n plant tissue 134 B. l F o l i a r elemental concentrations i n the boron experiment 135 B.2 F o l i a r elemental concentrations i n the copper experiment 136 B. 3 F o l i a r elemental concentrations i n the i r o n experiment 137 C. l S o i l p r o f i l e d e s c r i p t i o n of s i t e 1 138 C.2 S o i l p r o f i l e d e s c r i p t i o n of s i t e 2 140 C.3 S o i l p r o f i l e d e s c r i p t i o n of s i t e 3 142 C.4 S o i l p r o f i l e d e s c r i p t i o n of s i t e 4 144 C. 5 S o i l p r o f i l e d e s c r i p t i o n of s i t e 5 146 D. l Copper treatment: current-year growth (lodgepole pine) 148 D.2 Copper and nitrogen treatment: current-year growth (lodgepole pine) 149 D.3 Copper, i r o n and nitrogen treatment: current-year growth (lodgepole pine) 150 D.4 Copper treatment: second-year growth (lodgepole pine) 151 D.5 Copper and nitrogen treatment: second-year growth (lodgepole pine) 152 D.6 Copper, i r o n and nitrogen treatment second-year growth (lodgepole pine) 153 D.7 Control tree (lodgepole pine) 154 XV Appendix Page D.8 Iron treatment: current-year growth (lodgepole pine). 155 D.9 Iron and nitrogen treatment: current-year growth (lodgepole pine) 155 D.10 Iron treatment: second-year growth (lodgepole pine) . 156 D . l l Iron and nitrogen treatment: second-year growth (lodgepole pine) 157 D.12 Iron treatment: current-year growth (Douglas-fir) ... 158 D.13 Iron and nitrogen treatment: current-year growth (Douglas-fir) 158 D.14 Nitrogen treatment: current-year growth (lodgepole pine) 159 D. 15 Nitrogen treatment: current-year growth (Douglas-fir) 160 E. l Mean values f o r f o l i a r copper concentration (ppm) of lodgepole pine and Douglas-fir i n r e l a t i o n to treatments (main t r i a l ) 161 E.2 Mean values f o r f o l i a r a ctive i r o n concentration (ppm) of lodgepole pine and Douglas-fir i n r e l a t i o n to treatments (main t r i a l ) 164 E.3 Mean values f o r f o l i a r t o t a l i r o n concentration (ppm) of lodgepole pine and Douglas-fir i n r e l a t i o n to treatments (main t r i a l ) 167 E.4 Mean values for f o l i a r nitrogen concentration (%) of lodgepole pine and Douglas-fir i n r e l a t i o n to treatments (main t r i a l ) 170 xvi ACKNOWLEDGEMENTS I am deeply indebted to my supervisor, Dr. T.M. Ballard for his guidance and wise counsel which have contributed greatly to this research and also my study at U.B.C. I am also grateful to the other members of my Supervisory Committee: Dr. A.A. Bomke, Dr. L.M. Lavkulich, Dr. L.E. Lowe and Dr. J. Otchere-Boateng for their help and constructive criticism of this thesis. During the course of conducting this research, I was helped in a variety of ways by a number of other people and friends. In particular, I wish to acknowledge the help given by Ms. P. Carbis, Mr. J. Emanuel, Mr. D.G. Giles, Mr. F.M. Kelliher, Ms. J. Lansiquot, Mr. B. Von Spindler, Ms. E. Wolterson and Mr. B. Wong. I would like to acknowledge Dr. A. Kozak and Dr. H.E. Schreier for their advice in s t a t i s t i c a l analysis; Dr. R.E. Miller of the U.S. Forest Service for his advice on f o l i a r spray application; the B.C. Ministry of Forests for help in locating the research sites and providing seed; and the Natural Sciences and Engineering Research Council of Canada for financial support of the project. I would like to thank Universiti Pertanian Malaysia for granting me educational leave, and the Government of Malaysia for financial aid. Lastly, many thanks to my wife Fatimah for her love, support and encouragement. To my son, Azlan, who missed many happy moments with his father, I dedicate this thesis. 1 CHAPTER 1. GENERAL INTRODUCTION Plants are composed of a large number of chemical elements, several of which are categorized as "micronutrients" because they are required only in small amounts. These include boron, copper, iron, manganese, molybdenum and zinc. The essentiality of these micronutrients for healthy growth has been so well established for so many plant species that i t can be considered applicable for a l l . In forestry, micronutrient nutritional research i s s t i l l in i t s infancy. Our present knowledge on this subject is limited and fragmen-tary compared with that available either for cultivated plants or for macronutrients in forest trees. The problem one encounters in reviewing the literature on micronutrients in forest trees i s , therefore, mainly that of synthesizing the fragmentary reports from various fields of study including agriculture, horticulture, botany, ecology, biogeochemistry and forestry. One area of basic research that has gained some attention in forestry i s the determination of mineral requirements of various tree species. Most of these investigations, however, pertained to macro-nutrient requirements, with relatively very l i t t l e effort directed towards micronutrients. This is despite the fact that micronutrient deficiencies have been identified in forest situations in many parts of the world (Stone 1968). Perhaps this is partly because of the special experimental d i f f i c u l t i e s involved in the study of micronutrients (Fortesque and Marten 1969). 2 Another area of f o r e s t r y research that has attracted much attention i n recent years i s forest f e r t i l i z a t i o n . There are numerous reports on f e r t i l i z e r t r i a l s and operational f e r t i l i z a t i o n i n North America, Europe, A u s t r a l i a , New Zealand and the t r o p i c s . The majority of these, however, involved s o i l a p p l i c a t i o n of f e r t i l i z e r s and to some extent a e r i a l a p p l i -cation of granular macronutrient f e r t i l i z e r s ( M i l l e r and Fight 1979; DeBell 1981; Morrison 1981; Pulsford 1981). There i s very l i m i t e d i n f o r -mation on f i e l d f o l i a r a p p l i c a t i o n of l i q u i d f e r t i l i z e r s on forest trees. This method of f e r t i l i z e r a p p l i c a t i o n has been used i n a g r i c u l t u r e for a long time and has been an accepted p r a c t i c e i n supplying various micronutrients f o r many crops (Murphy and Walsh 1972; Traynor 1980). Some f o l i a r analysis work i n i n t e r i o r B r i t i s h Columbia l e d to inference of possible micronutrient d e f i c i e n c i e s i n some f o r e s t stands (Ballard 1981). In order to improve diagnosis of such problems and explore possible remedial measures, t h i s thesis research was c a r r i e d out i n the form of two complementary studies. The f i r s t study, reported i n Chapter 3, involves three greenhouse experiments on micronutrient requirements of lodgepole pine (Pinus contorta Dougl.). Chapter 4 reports findings from f i e l d experimentation on f o l i a r a p p l i c a t i o n of f e r t i l i z e r solutions to lodgepole pine and Douglas-fir (Pseudotsuga  menziesii [Mirb.] Franco). S p e c i f i c objectives of these two i n v e s t i g a -tions are given i n the respective chapters. A general review of l i t e r a t u r e i s given i n Chapter 2. 3 CHAPTER 2. LITERATURE REVIEW A• Nutrient Requirement Evaluations Evaluation of nutrient requirements of forest trees and identifica-tion of mineral deficiencies can be conducted by a number of different methods. Each has i t s own applications, merits, and drawbacks. Morrison (1974) reviewed five commonly employed methods for conifers. These include f e r t i l i z e r f i e l d t r i a l s , f e r t i l i z e r pot t r i a l s , visual symptoms, s o i l analysis and f o l i a r analysis. This section gives a general review of these methods, four of which ( f e r t i l i z e r f i e l d t r i a l , f e r t i l i z e r pot t r i a l , visual symptoms and f o l i a r analysis) were used in the two investigations reported in this thesis. 1. F e r t i l i z e r Field Trials Tamm (1964) affirmed that "no single method other than direct f i e l d experiments can give complete and reliable information on the nutrient status of a forest stand." He also pointed out i t might not always be economically feasible to conduct large scale f i e l d experiments. The use of single-tree plot technique suggested by Viro (1967) has been used to overcome this problem. It permits a large number of treatments to be tested, reduces costs, provides faster and reasonably reliable indica-tions of treatment responses (Gessel et a l . 1960; Viro 1967, 1970). If the danger of f e r t i l i z e r fixation in the s o i l exists, as in the case for micronutrients, the use of f o l i a r spraying is the preferred method (Murphy and Walsh 1972). 4 2. F e r t i l i z e r Pot Trials Pot t r i a l s or greenhouse techniques have been widely used in forest nutrition research because they allow the use of more complicated designs under less variable s o i l and climatic conditions. In addition, results could be obtained in a much shorter time and are generally less d i f f i c u l t to evaluate. Naturally there are several limitations to greenhouse studies. The main disadvantage is the problem of extrapolating results to f i e l d condi-tions. For instance, the nutrient requirements of seedlings are different from those of adult trees (Tamm 1964; Mead and Pritchett 1971). Despite the a r t i f i c i a l environment, much valuable information could be obtained for diagnostic purposes (Walker et a l . 1955; Will 1961; Ingestad 1979). For micronutrients, Bar rows (1959) indicated that because of the complexity of nutrient interactions in plants, the establishment of nutrient requirements and c r i t i c a l ranges of the micronutrients could be done only when a l l other essential elements are present in the plant i n the amounts and ratios appropriate for maximum growth. This condition can be best attained under controlled conditions with sand or solution culture, from which the complexities of s o i l are excluded. 3. Visual Symptoms The use of visual symptoms is perhaps the most direct method employed in plant nutrition research, and often gives the f i r s t indica-tions of any nutritional disorder. The advantages of this method reside in i t s simplicity and in the fact that laboratory f a c i l i t i e s are not required. Although visual symptoms can be useful, they have some 5 limitations. For instance, not a l l deficiencies show distinctive symptoms. Those of N, Ca, S, Fe or Mn tend to produce nonspecific general chloroses (Morrison 1974). If there are two or more deficiencies in a plant, the symptoms of one may mask those of the others, or the symptoms may not appear to be typical of any one element. Also, by the time visual symptoms have appeared, the supply of the element in the plant has reached the deficiency level and growth has already been reduced (Barrows 1959). Another limitation in using visual symptoms is what Zottl (1973) termed "hidden hunger." It is a situation where tree growth can be severely reduced without any clear visual symptom. It is therefore desirable to have a diagnostic tool to detect an adequate level of any essential element before i t drops to the point where visual symptoms begin to appear. 4. Soil Analysis The usefulness of s o i l chemical data for tree nutrient status interpretation and for prediction of nutrient supply to forest trees is limited (Armson 1973; van den Burg 1976; Khanna 1981). Khanna attributed this failure to technical and conceptual d i f f i c u l t i e s . The technical d i f f i c u l t i e s involve obtaining a representative s o i l sample. Variability in the distribution of tree roots and heterogeneity of forest soils make i t extremely d i f f i c u l t to collect samples that could represent the nutrient-supplying status of the entire s o i l volume. This is particular-ly c r i t i c a l with micronutrients because, some of them, such as boron, are very soluble and move readily through the s o i l whereas others, such as copper and zinc, are immobilized and tend to accumulate near the surface (Barrows 1959). 6 The conceptual problem involves the d i f f i c u l t y in determining the available or mobilisable forms of nutrients in nature. Total analysis for s o i l nutrients does not offer meaningful values for availability of any particular nutrient to the plant. Many extractants have been used to measure the 'available' portion of the s o i l nutrient, but the values obtained are seldom comparable (van den Burg 1976). For micronutrients, Cox and Kamprath (1972) stated that s o i l tests have often proved unreliable because of insufficient information about the chemistry of micronutrients in s o i l and absorption mechanisms by plant roots. These d i f f i c u l t i e s have led, in recent years, to a prefe rence for the use of fo l i a r analysis over s o i l analysis. 5. Foliar Analysis Foliar analysis is a technique that measures not the supply of nutrients available in the s o i l but an index of the amount taken up by the tree (Morrison 1974). It has been used to diagnose nutrient deficiencies (Ballard 1981; Carter ejt al^., 1984), in interpreting responses in f e r t i l i z e r t r i a l s (Richards and Bevege 1969; Lea et a l . , 1980), and to establish basic relationships between nutrient concentra-tion and supply (van den Driessche 1969; Swan 1972; Ingestad 1979). Basically, the use of fo l i a r analysis for the f i r s t two objectives mentioned above (diagnosis of nutrient deficiencies and interpretation of f e r t i l i z e r response) is based on the assumption that a quantitative relationship exists between plant growth and fo l i a r nutrient levels. This basic relationship i s referred to by Armson (1973) as a growth response curve. Figure 1 illustrates this relationship. When a nutrient is limiting growth, i n i t i a l input of that nutrient w i l l increase growth TISSUE NUTRIENT CONC. OF AN ELEMENT Figure 1. Generalized r e l a t i o n s h i p between 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 . 8 but a further increase in the supply of that nutrient tends to cause the growth to level off. Additional input beyond this point may cause a decline in growth. A central concept used for interpretation of this growth response curve is that there is a " c r i t i c a l range" (BC in Figure 1) or " c r i t i c a l percentage" (B in Figure 1) of each nutrient for each kind of plant (Macy 1936; Barrows 1959). Below this range (AB in Figure 1) trees w i l l likely show deficiency symptoms and w i l l respond substantially in increased growth to applications of the element in question. Above this range, (CD in Figure 1) there is luxury consumption, where no beneficial response to applications of the element would be expected. In Figure 1, DE represents the toxicity range. The " c r i t i c a l percentage" is defined as associated with 90 percent of maximum yield (Richards and Bevege 1970; Swan 1972). The use of f o l i a r analysis, hence the c r i t i c a l levels, to assess nutritional deficiencies is not free from limitations. In many instances, the c r i t i c a l ranges or levels were determined under controlled greenhouse conditions (Morrison 1974). It is therefore questionable whether the values obtained could be applied to actual f i e l d conditions where environmental and physiological factors may modify tree nutrient status (Leaf 1968, 1973). Armson (1973) also warned that the greatest unrelia-b i l i t y of comparative values may be expected when these values are extra-polated from one growing location to another or to stands at different stages of development. The d i f f i c u l t y is further aggravated in the case of micronutrients. One main limitation in using c r i t i c a l values for micronutrients is 9 associated with elemental i n t e r a c t i o n s (Watanabe et a l . , 1965). The d e f i c i e n c y and optimum ranges have been found to overlap because the response to one nutrient often depends upon the l e v e l of other nutrients (DeKock 1955, 1981). A second l i m i t a t i o n , as indicated by H i l l and Lambert (1981) i s that sometimes a C-shaped response curve e x i s t s betwen growth and micronutrients concentrations i n the f o l i a g e , compared to the c u r v i l i n e a r curve i n Figure 1. In t h i s case, the nutrient present i n very l i m i t i n g amount i n the plant may f i r s t show a decrease and then an increase i n percentage concentration with increase i n growth. This i s commonly associated with c e r t a i n micronutrients (Steenbjerg 1954). However, U l r i c h and H i l l s (1973) stated that one of the greatest values of f o l i a r analysis i s being able to prevent d e f i c i e n c i e s rather than correct them a f t e r they occur. These authors also mentioned that the c r i t i c a l concentration i s estimated best through the use of s o l u t i o n culture and to a lesser extent by s o i l culture or f i e l d experiment techniques. B. Expression of F o l i a r Nutrient Composition Nutrient composition i n plant tissue i s commonly expressed as concentration on a dry mass basis, for example, percentage of dry weight of the tissue (for macronutrients), or i n parts per m i l l i o n (for elements present i n small amounts). It has also been expressed i n terms of absolute content i n the tissue (e.g. gram per shoot or per needle). Leaf (1973) noted that the terms concentration and content have often been used synonymously, even though the d i s t i n c t i o n between the two should be quite obvious (Farhoomand and Peterson 1968). 10 Concentration has been widely used because of the assumption that concentration alone can sufficiently indicate whether a particular element is at a deficient, adequate or toxic level in the plant (Farhoomand and Peterson 1968). Van den Driessche (1974) mentioned that nutrient measurements should be independent of growth parameters such as weight, i f the objective is to infer whether a nutrient is growth-limiting. Generally, i f a particular element that is limiting growth i s added in a f e r t i l i z e r treatment, an increase in the concentration of the element in the tissue occurs. This relationship is regarded as positive and unequivocal (Figure 1). However i t is possible that a negative relationship may occur (Steenbjerg 1954), as demonstrated by Ebbel (1972). This has been described as "dilution effect" (Munson and Nelson 1973) or "Steenbjerg effect", where a decrease in concentration occurs due to a relatively large increase in size or dry matter production which dilutes the nutrient in the plant tissue. This may confuse diagnostic interpretations. Content, on the other hand, may increase because of the continuous uptake. Therefore, expressing f o l i a r nutrient data on a content (uptake) basis would help overcome diagnostic imprecision due to the dilution effect (Farhoomand and Peterson 1968). Another method of expressing nutrient composition, as suggested by Stachurski and Zimka (1975), is in terms of content per unit of leaf area. The supporting argument is that expressing nutrient composition on a dry weight basis may confound interpretations because of major changes in tissue dry weight resulting from fluctuations in stored carbohydrates (Bradbury and Malcolm 1978). Gholz (1978) and Smith et^al. (1981) found 11 this method to be superior to expression on a dry weight basis. It i s , however, uncertain whether dilution effect would be reduced i n using leaf area (van den Driessche 1974). The fourth method of examining f o l i a r nutrient data is to use c r i t i c a l and optimum nutrient ratios (Ingestad 1970). The basis for using ratios i s that f o l i a r composition i s associated with an ideal equilibrium among elements in the plant tissue (Bonneau 1973). This notion has been supported by van den Driessche (1974) who noted that maximum yield can only be achieved when a l l nutrients are at optimum concentration and balance. Interactions between nutrients can also confound diagnostic inter-pretations. According to Olsen (1972), the effects of nutrient inter-action could either be mutual (synergism) or reciprocal (antagonism). In a synergistic interaction, the uptake or translocation of one element by a plant i s stimulated by another element. The opposite takes place in antagonistic interaction. Interations, therefore, tend to militate against direct interpretation of nutrient concentrations or absolute content. This has led to the use of nutrient ratios to rationalize such interactions. Some of the commonly used ratios in forest nutrition research are N/S, N/P, N/K, K/Ca, Ca/Mg, and P/Al (Leyton 1958; Tamm 1964; Waring 1972; van den Driessche 1974). The methods of expressing nutrient composition mentioned above used techniques that measure total concentration or content in the plant tissue. Leece (1976) cr i t i c i z e d this approach because i t does not distinguish the physiologically active from the inactive fraction of a particular nutrient element under study. This concept was already being 12 applied to "active" i r o n i n f r u i t trees a half-century ago by Oserkowsky (1933). He defined a c t i v e i r o n as that form of i r o n that i s a c t i v e i n c h l o r o p h y l l formation. Zech (1970) applied t h i s concept to d i f f e r e n t i a t e between c h l o r o t i c and normal trees of Scots pine (Pinus s i l v e s t r i s L . ) . He found that the a c t i v e i r o n l e v e l i n c h l o r o t i c needles was less than that of green ones though the t o t a l i r o n content was sometimes higher i n c h l o r o t i c needles. Hence, t o t a l i r o n i n the f o l i a g e i s sometimes not i n d i c a t i v e of i r o n d e f i c i e n c y . The present study employs four of the f i v e methods mentioned to express nutrient composition. These are concentration, content (mg element per 100 needles), some nutrient r a t i o s , and active i r o n . C. Nutrient Requirements of Conifers Some nutrient concentration values for a selected number of coni-ferous species are summarized i n Table 1. More detai l e d reviews were given by Stone (1968) and Morrison (1974). A more recent review f o r coniferous species i n B r i t i s h Columbia was done by B a l l a r d and Carter (1983), but as cautioned by the authors, many of the values given are not s p e c i e s - s p e c i f i c . Some of the values obtained i n the l i t e r a t u r e were used i n the text of t h i s thesis f or comparative purposes. The p o t e n t i a l l i m i t a t i o n s discussed i n previous sections were recognized when i n t e r -preting and extrapolating these values. 13 TABLE 1. F o l i a r nutrient values u s e f u l i n i n d i c a t i n g nutrient status of c o n i f e r s . Deficient Type of ( c r i t i c a l culture value/range) Adequate High Reference Pinus contorts Sand Pinus contorta F i e l d Pseudotsuga • e n z l e s l l F i e l d NITROGEN, I dry weight 1.20 - 1.70 1.7 - 3.0 1.0 1.50 1.0 1.40 >3.0 Swan 1972 Everard 1973 Everard 1973 Pinus contorta Sand Plnua contorta F i e l d Pseudotsuga n e n z l e s l l F i e l d PHOSPHORUS, X dry weight 0.10 - 0.17 0.17- 0.40 0.12 - 0.15 0.15 0.14 0.20 >0.40 Swan 1972 Everard 1973 Everard 1973 Pinus contorta Sand Pinus contorta F i e l d Pseudotsuga menzlesll F i e l d POTASSIUM, I dry weight 0.30 - 0.50 0.50 - 1.10 0.40 - 0.50 0.50 0.50 0.70 >1.10 Swan 1972 Everard 1973 Everard 1973 Pinus contorta  Pseudotsuga menzlesll Sand CALCIUM, I dry weight 0.06 - 0.06 0.08 - 0.30 >0.30 Swan 1972 0.25 Ballard and Carter 1983 Pinus contorta  Pseudotsuga menzlesll Sand MAGNESIUM, I dry weight 0.07 - 0.09 0.09 - 0.16 >0.16 Swan 1972 0.10 Ballard and Carter 1983 Pinus s l l v e s t r l s Solution SULPHUR, Z dry weight 0.04 - 0.15 0.15 - 0.20 Ingested 1960 BORON, ppm Pinus ponderosa F i e l d - 14 - 135 - Parker 1956 Pinus patula F i e l d 7 - 1 8 - - V a i l et a l . 1961 Pinus pinaster F i e l d 8 16 - Stone and W i l l 1965 Pinus radiata F i e l d 8 - - W i l l 1971 Pinus radiata F i e l d 5-7 - - Windsor and K e l l y 1972 Pinus realnoaa F i e l d - - 54-75 Neary et a l . 1975 Pinus radiata Pot ( s o i l ) 6 - - Snowdon 1982 Pinus taeda Nursery 1.8 - 4.2 - - Stone et a l ^ 1982 Pinus s l l v e s t r l s F i e l d 4 - - Aronsson 1983 14 IABLE 1. (cont'd) Deficient Type of ( c r i t i c a l culture value/range) Adequate High Reference COPPER, ppm Picea sitchensis Nursery 2.3 - 2.8 7.8 -10.8 - fienzian and Warren 1956 Pinus ponderosa F i e l d - 2.5 - 7.9 - Parker 1956 Pseudotsuga menziesii Nursery 2.6 4.5 - Oldenkamp and Smllde 1966 Pinus radiata F i e l d 1.0 3.0 - Rulter 1969 Pinus radiata F i e l d 3.0 - - W i l l 1971 Pinus e l l i o t t i S o i l (pot) - - 14-30 van Lear and Smith 1972 Pinus radiata Nursery 2.0 - - Knight 1975 Pseudotsuga menziesii F i e l d 4 - - S t r u l l u and Bonneau 1978 Pseudotsuga menziesii F i e l d 1.5 - - Binns e_t a_l. 1980 Pseudotsuga menziesii Vermiculite 1.7 - 2.6 4 - Lambert and Weidensaul 1982 IRON, ppm Pinus s i l v e s t r i s Solution 50 50 - 70 - Ingestad 1960 Pinus s i l v e s t r i s F i e l d 50 - - Zech 1970 Pinus radiata F i e l d 34 - - Ruiter 1983 MANGANESE, ppm Pinus ponderosa F i e l d - 38 -102 - Parker 1956 Picea abies F i e l d 4 - 2 0 20 - Ingestad 1958 Pinus radiata F i e l d 20 - 30 - - Woods 1983 ZINC, , ppm Pinus radiata F i e l d 10 - - Ruiter 1972 Pinus radiata Nursery 15 15 - 50 - Knight 1975 Pinus radiata F i e l d 10 - 15 - - McGrath 1978 Pinus radiata F i e l d 10 - 12 - - Woods 1983 15 CHAPTER 3. GREENHOUSE EXPERIMENTS A. Introduction There i s very limited information from the literature on various aspects of micronutrient nutrition of forest trees. The extensive review by Stone (1968) on this subject emphasized this lack of information, but l i t t l e progress has been achieved since Stone published his review. As evident from the literature review section, there has been no research reported in Canada on the determination of the c r i t i c a l levels of micro-nutrients for any of the coniferous species. In fact, there i s no published information on this subject for lodgepole pine, which is the most wide-ranging of the American pines (Critchfield 1980). This chapter reports findings from a greenhouse study conducted with lodgepole pine as the test species. The main objective of the experiments was to bracket the c r i t i c a l levels of boron, copper, total iron (Fe) and active iron (AFe) for lodgepole pine. B. Methods and Materials 1. Experimental Design Essentially the study consisted of three separate experiments. In each of these experiments, the supply of one of the three micronutrients (boron, copper and iron) was given at three levels and associated with two levels of nitrogen. The other eleven essential nutrients were supplied at levels known to be sufficient for optimum growth. 16 The reason f o r supplying nitrogen at two l e v e l s (low and adequate) i s because of the assumption that there i s a ph y s i o l o g i c a l i n t e r a t i o n between nitrogen and some micronutrients. Applications of nitrogen f e r t i l i z e r s have been shown to induce and/or accentuate d e f i c i e n c i e s of boron (Moller 1983) and copper (Ruiter 1969) i n Pinus spp. growing on s o i l s marginal i n terms of a v a i l a b i l i t y of these n u t r i e n t s . Both nitrogen and i r o n play an e s s e n t i a l r o l e i n chlo r o p h y l l formation (Mengel and Kirkby 1982). The c r i t i c a l l e v e l cannot be determined p r e c i s e l y from t h i s study design because there are too few data points f o r precise estimation of a response curve. When maximum observed growth i s associated with the intermediate nutrient supply l e v e l , data associated with t h i s l e v e l can be used f o r preliminary approximation of " c r i t i c a l " values. Subsequent research w i l l be needed f o r confirmation and/or further refinement of such estimates. Each experiment b a s i c a l l y consisted of a 3 x 2 f a c t o r i a l treatment combination r e p l i c a t e d four times. Each r e p l i c a t e was represented by one pot, giving a t o t a l of 72 pots for the three experiments. The treatments were arranged i n a completely randomized design. Experimental duration was s i x months. 2. Pot Arrangement The p o s i t i o n of each pot on the bench was rotated each time nutrient a p p l i c a t i o n was made. This was to reduce e f f e c t s of environ-mental differences (for example, short wave r a d i a t i o n and temperature differences) along the greenhouse bench. 17 3. Greenhouse Environment The greenhouse was cooled during the summer months with automatic ventilators, and steam-heated during the f a l l . The temperature range was from 14°C to as high as 35°C during the summer. Relative humidity was about 75 percent. At the top of the seedlings under natural lighting in mid-March, quantum flux density in the 0.4 to 0.7 \im wave band was only 150-200 umol m~^ s--'-, as measured with a Quantum Sensor. It was necessary to increase the light intensity to about 300 pmol m~^s~^ for rapid and continuous growth of seedlings. This was done with supplemen-tary lighting from fluorescent lamps. A 16-hour photoperiod was main-tained for the entire duration of the study. 4. Preparation and Maintenance of Growth Medium Pure white sand (trade name "20-30 V S i l i c a Ottawa Sand") was used as the growth medium. The sand was acid washed, using the method by Hewitt (1966), modified slightly because of high impurities. The proce-dure involved submerging the sand i n a mixture of 2N HCI and 1 percent oxalic acid for one week. This was repeated for three consecutive weeks, with fresh acid for each weekly washing. The sand was then washed with demineralized water to remove any of the surplus acid from the growth medium. A total of 35 washings for each container of 30 kg sand was necessary to raise the pH from 0.5 to 5.5, which was a suitable pH for seed germination. Analysis of the f i n a l leachate showed no trace of the micronutrient metal elements to be tested, and negligible amounts of some macronutrients (Table 2). This laborious but thorough washing procedure 18 TABLE 2. Chemical analysis of the leachate from the sand used as the growth medium. Element Concentration K 0.02% Ca 0.01% Mg 0.0% Cu 0.0 ppm Fe 0.0 ppm Mn 0.0 ppm Zn 0.02 ppm was necessary to prepare a satisfactory batch of pure sand crucial for the micronutrient studies. About 4 kg of the sand was placed in each of the 8-liter plastic pots. Drainage holes at the bottom of each pot were covered with a piece of polyethylene cloth, to prevent any loss of sand. Each pot was placed in a 10-inch diameter plastic tray, used to collect leachate for weekly pH measurements. Because the sand and nutrient solution were essentially unbuffered, large and rapid pH changes could occur. It was found that, after every three nutrient applications, the pH dropped from about 5.5 to 2.0. This pH d r i f t due to formation of acids in the sand occurred throughout the study period of six months. It was necessary to raise the pH back to 5.5, and this was done by flushing each pot with at least six l i t r e s of demineralized water once every week. This washing also tended to remove algae that grew on the sand culture. 19 The moisture content of the sand was maintained at 10 percent by weight. At this level, the water content i s approximately two-thirds of f i e l d capacity (Allen 1968). The pots were occasionally weighed as the seedlings grew bigger, to check that the water content was at this level. It was necessary to increase the frequency of watering during the summer months. 5. Germination and Thinning The seeds were provided by the Silviculture Branch, British Columbia Ministry of Forests. These seeds were collected from two locations in the Prince George region: Willow River area (Seedlot No. 2093) and Vanderhoof area (Seedlot No. 2313). Prior to sowing, the seeds were placed in demineralized water for imbibition and then were placed in a ring of shallow indentations made on the surface of the wet sand. Pots were seeded on March 18, 1982. Seeds from both seedlots were sown i n each pot but separated by a 2-cm plastic divider placed across the center of the pot. One hundred seeds were sown in each pot. A plastic cover was placed over the pots during the germination period in order to maintain high humidity. Emergence of about 85 percent was achieved after eight days. Five thinnings were done during the duration of the experiment. Details are outlined in Table 3. At the end of six months of growth, satisfactory inducement of deficiences was very noticeable and sufficient biomass was produced from each replicate (pot) for chemical analysis. 20 TABLE 3. Thinning frequency, dates and number of seedlings remaining after each thinning. Number of Number of remaining thinning Date seedlings per pot 1 Ap r i l 18 40 2 May 18 24 3 June 18 18 4 July 18 16 5 August 18 14 Final harvest September 18 6. Composition of Nutrient Solutions The three experiments were designated as boron, copper and iron experiments, each having six treatments (three levels of the test micro-nutrients and two levels of nitrogen). The composition of the nutrient solution was based on that used by Swan (1972). The solution concentra-tions of the "manipulated" elements (B, Cu, Fe and N) are given in Table 4. In each of the three separate experiments, the composition of the standard nutrient solutions was the same for the non-manipulated elements. Tables 5 and 6 give solution composition information about these, for the lower (10 ppm) N and higher (100 ppm) N treatments, respectively. 21 TABLE 4. Concentration of manipulated elements in nutrient solutions. Test element Nutrient level (ppm) 1 2 3 Boron 0 0.04 0.40 Copper 0 0.002 0.02 Iron 0 0.05 5.0 Nitrogen 10 100 7. Nutrient Application The nutrient solutions were prepared in demineralized water. Before application, the pH of the solution was adjusted to pH 5.5 with 0.025N NaOH. Treatment was done two weeks after sowing and subsequently once every two days for the entire duration of the experiment. The nutrient solution was applied directly to the sand surface, avoiding contact with the upper portions of the seedlings. The volume applied was one l i t r e per pot. Supplementary watering was done only in July when It was extremely warm and necessitated the addition of 500 mL of deminer-alized water to each pot the day following treatment application. Preparation and application of nutrient solutions were a l l done in plastic measuring cylinders and containers. A l l work was manually done and every possible effort was taken to avoid contamination. 22 TABLE 5. Concentration and source of elements in standard nutrient solution for level 1 nitrogen supply (10 ppm). Element Element ppm Source Source Associated Element mg/L element ppm N 4.969 NH4C1 2.795 Ca(N03)2.4H20 2.238 KNO3 18.98 23.56 16.16 Cl Ca K 12.57 4.0 6.24 10.0 KH2PO4 43.90 K 12.61 K 6.24 12.61 56.15 KNO3 KH2P04 KC1 107.25 Cl 51.07 Mg 50.0 MgSO4.7H20 506.72 65.98 Ca 4.0 36.0 Ca(N03)2.4H20 CaCl 2 99.68 Cl 63.89 65.98 2.87 MgS04.7H20 FeS04.7H20 Cl 12.57 51.07 63.89 NH4CI KC1 CaCl 2 Fe 5.0 FeS04.7H20 24.90 2.87 Mn 0.20 Cu 0.02 MnCl2.4H20 CuS04.5H20 0.72 0.079 Zn 0.05 0.40 Mo 0.03 ZnS04-7H20 H3BO3 Na2Mo04.2H20 0.22 2.29 0.071 23 TABLE 6. Concentration and source of elements in standard nutrient solution for level 2 nitrogen supply (100 ppm). Element Element ppm Source Source Associated Element mg/L element ppm N 49.69 NH4C1 27.95 Ca(N03)2.4H20 22.38 KNO3 189.8 235.6 161.6 CI 125.7 Ca 40.0 K 62.39 10.0 KH2PO4 43.9 K 12.61 K 62.39 Mg 50.0 Ca 40.0 KNO-3 12.61 KH2P04 MgS04.7H20 Ca(N03)2.4H20 506.72 65.98 65.98 MgS04.7H20 2.87 FeS04.7H20 CI 125.7 NH4CI Fe 5.0 Cu Zn B • Mo 0.40 FeS04.7H20 Mn 0.20 MnCl2.4H20 0.02 CuS04.5H20 0.05 ZnS04.7H20 H3BO3 0.03 Na2Mo04.2H20 24.9 0.72 0.079 0.22 0.29 0.071 2.87 24 8. Harvesting, Sample Measurement and Preparation Visual symptoms for a l l 18 treatments were recorded before harvesting. A l l seedlings were harvested by cutting off the stems at sand level. The seedlings in each pot from the two different seedlots (provenances) were processed separately, except for the roots, which were impossible to separate. A l l seedlings were thoroughly washed with demineralized water after harvest. The needles were then stripped from the stems and heights were measured on five seedlings per provenance per replicate. Needles, stems and roots were dried at 70°C in a forced-draft oven. Drying of needles took at least 10 hours, unt i l they could be snapped cleanly into two when bent. (This was the criterion used to estimate whether samples were dry enough for efficient grinding). It took about 24 hours to dry the stems and roots to get to a constant weight. The following weights were taken: stem, root, total foliage and one hundred (randomly selected) needles. Needle, stem and root samples were ground separately in a Waring blender stainless steel cup, and kept in airtight screw-cap plastic bottles. Before chemical analyses, sufficient portions of each sample were re-dried i n the oven at 65°C for three hours, cooled in a desic-cator, and the required amount was then weighed for analysis. 9. Chemical Analysis The wet digestion method of Parkinson and Allen (1975), slightly modified by Ballard (1981), was employed to prepare foliage, stem and 25 root samples for the determination of total N, P, K, Ca, Mg, Al, Cu, Fe, Mn and Zn. Total N and P were simultaneously analysed on the original digest by the Technicon Autoanalyzer II. Potassium, Ca, Mg, Al, Cu, Fe, Mn and Zn concentrations in the digest were determined by atomic absorp-tion spectophotometry. Details of the procedure are outlined in Appendix A . l . The values for Cu and Fe were noticeably low, especially for Cu where the concentration In the digest approached the instrument's sensitivity limit. Therefore foliage samples were re-analysed for these two elements using a more accurate and sensitive method involving HNO3 digestion. Details of this procedure proposed by Ballard (personal communication^-) are described in Appendix A.2. The values obtained by this method were used in this thesis. The concept that a fraction of the total iron i s metabolically active (Oserkowsky 1933) warranted that this form of iron to be deter-mined. Appendix A.3 outlines Oserkowsky's procedure for active iron determination as modified slightly by Ballard (1981). Boron was determined by the azomethine-H colorimetric procedure of Wolf (1971, 1974), as modified by Gaines and Mitchell (1979). This procedure involves dry ashing; this element can be volatilized during wet ashing (Jones and Steyn 1973). Determination of total sulphur was done using a Fisher Model 475 Sulphur Analyzer. The procedures used were those in the manufacturer's manual, with some modifications as described by Lowe and Guthrie (1981). T.M. Ballard. Professor, Faculty of Forestry/Department of Soil Science, University of British Columbia. 10. S t a t i s t i c a l Analysis Comparison between provenances was not an objective of t h i s thesis research. Therefore, the data obtained f o r the two provenances were pooled to give a single value f o r each variable measured for each r e p l i c a t e . Results of stem and root analysis are not discussed i n t h i s t h e s i s . The assessment of c r i t i c a l l e v e l s f or B, Cu, AFe and Fe was based on the assumption that the c r i t i c a l l e v e l on the growth response curve (Figure 1) i s at a point where 90 percent of maximum growth occurs (Richards and Bevege 1970; Swan 1972). A l l data were subjected to analysis of variance and means were separated by the Duncan's New Mu l t i p l e Range t e s t . C. Results and Discussion 1. Boron Experiment (a) V i s u a l Symptoms Complete withdrawal of boron (B^) f o r both supply l e v e l s of nitrogen (N^ and N 2) produced severe reduction i n seedling growth. Growth reduction was more severe at the lower nitrogen supply (N^) i n d i c a t i n g nitrogen d e f i c i e n c y as w e l l . The stems were t h i n and crooked Needles were short and s t i f f with bronze or orange c o l o r a t i o n . Terminal needles formed a c l u s t e r due to adhesion of needle t i p s to each other with r e s i n . These symptoms are quite s i m i l a r to those described by Snowdon (1982) f o r radiata pine seedlings and Stone et a l . (1982) for slash pine i n the nursery, both s u f f e r i n g from boron de f i c i e n c y . 27 There was no appreciable difference i n needle colour between the higher l e v e l s of boron ( B 2 and B 3 ) , except that the needles were s l i g h t l y greener at the N 2 than the l e v e l s . Seedling height and biomass were d i s t i n c t i v e l y highest for treatment B3N2. The above descriptions are i l l u s t r a t e d i n Figures 2, 3 and 4. Although seedling growth was greater with B2N2 than with B2^i, such deficiency symptoms as needle twisting and d i s c o l o r a t i o n seemed more severe with B2N2- 'At t h i s same l e v e l of boron supply, f o l i a r boron concentration at the N 2 l e v e l was s i g n i f i c a n t l y lower (P <^  0.05) than at the l e v e l (Table 7). There was a highly s i g n i f i c a n t difference (P <^  0.01) i n f o l i a r nitrogen composition between the two supply l e v e l s of N (Table 8). F o l i a r nitrogen at the and N2 supply l e v e l s were about 1 and 2 percent, r e s p e c t i v e l y (Appendix B . l ) . The above f i n d i n g was consistent with the hypothesis that better s o i l nitrogen status, allowing more biomass production, r e s u l t s i n imposition of a greater demand f o r boron (Stone 1968). Such a r e s u l t has important p r a c t i c a l implications i n B r i t i s h Columbia where nitrogen d e f i c i e n c y i s frequently serious and nitrogen f e r t i l i z a t i o n i s often contemplated. On s i t e s where no boron deficiency problems have previously been evident, nitrogen f e r t i l i z e r might induce boron deficiency, i f the s o i l s boron supply has been marginal. In Sweden, Moller (1983) reported the occurrence of induced boron deficiency i n Scots pine when f e r t i l i z e d with nitrogen. Figure 2 . Lodgepole pine seedlings without boron supply. 29 Figure 3. Differential growth response of lodgepole pine seedlings to increasing boron supply at low nitrogen level. Figure 4. Differential growth response of lodgepole pine seedlings to increasing boron supply at high nitrogen level. TABLE 7. Foliar boron composition, mass of 100 needles and seedling height. Treatment Foliar B B N (ppm) Concentration (ppm) Content (mg/100 needles) Mass of 100 needles (g) Average seedling height (mm) 0 10 (B]_N]_) 11.7a (+1.68) 0.01a (+0.0001) 1.06a (+0.27) 91a (+1.13) 0.04 10 (B 2Ni) 43.4b (+5.42) 0.05b (+0.001) 1.16a (+0.29) 100a (+1.03) 0.40 10 (B 3N!) 107.3d (+7.72) 0.13c (+0.03) 1.22a (+0.25) 98a (+0.95) 0 100 (BiN 2) 7.3a (+3.04) 0.01a (+0.005) 2.01b (+0.26) 141b (+1.33) 0.04 100 (B 2N 2) 15.5a (+2.05) 0.04b (+0.005) 2.14b (+0.17) 158c (+0.79) 0.40 100 (B 3N 2) 85.5c (+9.83) 0.18d (+0.03) 2.03b (+0.14) 153c (+1.22) In each column, means with a different suffix are significantly different at the 5-percent level. Numbers in parenthesis are standard deviations. 32 TABLE 8. Analysis of variance of f o l i a r nutrient composition with varying supply levels of boron and nitrogen. Source Degrees of of N P K Ca Mg S Al B Cu AFe Fe Mn Zn variation freedom Boron 2 * ns ** ns ** ** * ** ns ns ns ** ** Nitrogen 1 ** ns ** ** ** ns ** ** * ** ** ** ** Boron x 2 ns ns ns ns * ns * ** ns ns ns ns ns Nitrogen Error 42 Total 48 > ns = Significant at the 0.05 and 0.01 levels, respectively. Not significant. 33 (b) C r i t i c a l Level The estimation of the c r i t i c a l level for boron was done at the higher nitrogen supply (100 ppm N) where boron was the only limiting element. Highest observed one hundred needle mass and height were associated with a f o l i a r boron concentration and content of 15.5 ppm and 0.04 mg/100 needles, respectively (Table 7). If these growth data repre-sent the growth response plateau of Figure 1, 90 percent of maximum growth would correspond to f o l i a r boron values of 7.3 ppm and 0.01 mg/100 needles (Table 7). Rounding off these values, the c r i t i c a l boron concen-tration could be assumed to be 7 ppm and the c r i t i c a l range from 7 to 16 ppm B. Figure 5 illustrates the relationships between f o l i a r boron and growth, where the latter i s expressed as either mass of 100 needles or seedling height. The above concentration values are within the range reported by Vail et a l . (1961), Stone and Will (1965) and Will (1971) on other pine species under f i e l d conditions. The value obtained i n this study might be used as an approximate and preliminary guideline to assess the boron status of lodgepole pine growing under f i e l d conditions. Actual f i e l d experimentation is necessary, however, to confirm or refine this guide-line • 2. Copper Experiment (a) Visual Symptoms There was slight difference in needle colour among the three copper treatments. Some degree of needle tip burn and chlorosis was 34 2 0 0 1 I50H SEEDLING HEIGHT (mm) l(XH N 2 N, N, N 2 5 0 ' — i 4 0 CONC. 8 0 (ppm) — i 120 0 0 . 0 6 0.12 0.18 CONTENT (mg) FOLIAR BORON Figure 5. Relationship between f o l i a r boron composition and seedling growth (lodgepole pine). 35 observed on seedlings that received low nitrogen but no copper (C^N^). This is probably due to both copper and nitrogen deficiencies. The most pronounced symptoms where copper was the only limiting element (C]^) were dark blue-green coloration, drooping and long tender needles, and bushiness. There was also a marked depression in height growth and biomass production. These symptoms are identical to those described by Binns et^ a l . (1980) for copper deficiency i n Sitka spruce and Scots pine. One of the functions of copper in plants is in c e l l wall metabo-lism, especially for lignin synthesis (Bussler 1981). Inhibition of li g n i f i c a t i o n due to copper deficiency w i l l lead to bending and loss of plant vigour. This probably explains drooping and soft needles observed in this experiment. Figures 6 to 8 illustrate the differential visual symptoms explained above. Foliar nutrient composition for this experiment is given in Appendix B.2. There was a highly significant difference (P £ 0.01) in fo l i a r nitrogen between the two levels of nitrogen supply (Table 10). Average f o l i a r nitrogen at the and N 2 levels were about 1.1 and 2.0 percent, respectively. Foliar copper concentration at the C3N2 level (at which copper and nitrogen were supplied in adequate amounts) was significantly lower than at the CgN^ level (Table 9). As in the case of boron, this result has important practical implications in that nitrogen f e r t i l i z a t i o n might induce copper deficiency in areas where no copper deficiency problems have previously been evident. Ruiter (1969) found that low copper concentrations in radiata pine foliage appeared to have been nitrogen-induced. 36 Figure 6. Lodgepole pine seedlings without copper supply. Figure 7. Differential growth response of lodgepole pine seedlings to increasing copper supply at low nitrogen level. 38 Figure 8- Differential growth response of lodgepole pine seedlings to increasing copper supply at high nitrogen level. TABLE 9. Foliar copper composition, mass of 100 needles and seedling height. Treatment Foliar Cu Cu N (ppm) Concentration (ppm) Content (mg/100 needles) Mass of 100 needles (g) Average seedling height (mm) 0 10 (ClNi) 1.1a (+0.48) 0.001a (+0.0001) 1.24a (+0.15) 99a (+1.04) 0.002 10 (C 2Ni) 2.5bc (+1.08) 0.003ab (+0.001) 1.08a (+0.27) 102a (+1.33) 0.02 10 (C3N1) 5.6d (+1.73) 0.007c (+0.002) 1.24a (+0.15) 121b (+1.02) 0 100 (CiN 2) 0.9a (+0.60) 0.002a (+0.001) 1.78b (+0.27) 128b (+1.47) 0.002 100 (C 2N 2) 1.8ab (+1.57) 0.004b (+0.003) 2.15c (+0.25) 161c (+1.01) 0.02 100 (C 3N 2) 3.0c (+0.63) 0.007c (+0.001) 2.33c (+0.14) 171c (+0.86) In each column, means with a different suffix are significantly different at the 5-percent level. Numbers in parenthesis are standard deviations. 40 TABLE 10. Analysis of variance of f o l i a r nutrient composition with varying supply levels of copper and nitrogen. Source Degrees of of N P K Ca Mg S Al B Cu AFe Fe Mn Zn variation freedom Copper 2 ** ** ** ** ** ns ns ns ** ** ** * ns Nitrogen 1 ** ** ** ** ** ** ** ** ** ** ** ** ** Copper x 2 ** ** ns * ** ** ns ns * ns * ** * Nitrogen Error 42 Total 47 *, ** = Significant at the 0.05 and 0.01 levels, respectively, ns = Not significant. 41 (b) C r i t i c a l Level Table 9 shows seedling growth response to varying levels of copper and nitrogen supply. At the N 2 (100 ppm N) level, biomass production and seedling height were highest when fo l i a r copper concentration was 3.0 ppm (or 0.007 mg Cu/100 needles). These relationships are depicted in Figure 9. Ninety percent of the maximum observed biomass and height growth was attained by seedlings having an average of 1.8 ppm Cu (0.004 mg Cu/100 needles) in their foliage. Rounding off these values, the c r i t i c a l copper concentration could be assumed to be 2 ppm and the c r i t i -cal range from 2 to 3 ppm Cu i f the maximum observed level approximates the actual maximum growth. The above concentration values are consistent with the findings of other investigators for conifers (Oldenkamp and Smilde 1966; Ruiter 1969; Strullu and Bonneau 1978; and Lambert and Weidensaul 1982). 3. Iron Experiment (a) Visual Symptoms The most obvious symptoms when iron was completely withdrawn were general chlorosis, long yellow needles, stunted bud development and severe retardation in height growth. When the seedlings were three months old, the needles turned to a whitish colour which is an indication of extreme iron deficiency ( H i l l and Lambert 1981). This symptom did not persist and the needles turned yellow when seedlings were five months old. With the F2N^ treatment (0.05 ppm Fe + 10 ppm N) the needles 42 3.0-1 50 H 1 1 H 1 1 0 3 6 0 0.005 0.01 CONC. (ppm) CONTENT (mg) FOLIAR COPPER Figure 9 . Relationship between f o l i a r copper composition and seedling growth (lodgepole pine). 43 showed bright yellow discoloration. The above descriptions are illustrated in Figures 10 to 12. Foliar nitrogen at the N 2 supply level was significantly higher (P < 0.01) than the N]_ level (Table 12, Appendix B.3). At the higher level of iron supply (F3), both total and active iron were s i g n i f i -cantly lower at the N 2 than level (Table 11). It appears that an increase i n fo l i a r nitrogen due to increased nitrogen supply caused a substantial decrease i n fo l i a r iron. Even though the actual f o l i a r iron values at the F 3N 2 level were relatively high (about 146 ppm), this finding implies an important practical consideration. If this decreasing trend in f o l i a r iron continues, repeated or high rate of nitrogen f e r t i l i z a t i o n might eventually induce iron deficiency. (b) C r i t i c a l Level The highest biomass and seedling height for the high nitrogen treatment (100 ppm N) were associated with f o l i a r total and active iron concentrations of 146.3 and 147 ppm, respectively (Table 11, Figures 13 and 14). Ninety percent of this maximum growth was attained by seedlings having 44.0 ppm total iron and 31.6 ppm active iron in their foliage. Rounding off these values, the c r i t i c a l concentration of total and active iron could be assumed to be 44 and 32 ppm, respectively. The concentra-tions associated with maximum growth are very high, and may well repre-sent "luxury" levels. Thus further study may be needed to estimate the c r i t i c a l range. The c r i t i c a l concentration estimates are highly compara-ble to those reported by Ingestad (1960) for total iron, and Zech (1970) for active iron i n Scots pine. Zech found that green needles had about 44 Figure 10. Lodgepole pine seedlings without iron supply. 45 Figure 11. Differential growth response of lodgepole pine seedlings to increasing iron supply at low nitrogen level. 46 F>N1 F l r j 2 . FiSZ F i g u r e 1 2 . D i f f e r e n t i a l growth response of l o d g e p o l e pine s e e d l i n g s to i n c r e a s i n g i r o n s u p p l y a t h i g h n i t r o g e n l e v e l . TABLE 11. Total and active iron composition, mass of 100 needles and seedling height. Treatment Fe N (ppm) Total f o l i a r Fe Concentration Content (ppm) (mg/100 needles) Active Fe Concentration Content (ppm) (mg) Mass of 100 needles (g) Average seedling height (mm) 0 10 (FiNi) 48.3a (+6.54) 0.05a (+0.01) 37.7a. (+13.17) 0.04a (+0.01) 1.09a (+0.16) 108a (+1.16) 0.05 10 (F 2N L) 81.4a (+30.31) 0.08a (+0.04) 54.8a (+14.33) 0.05a (+0.01) 1.01a (+0.29) 106a (+1.09) 5.0 10 (F 3Ni) 359.4c (+126.89) 0.47c (+0.17) 351.1c (+120.16) 0.46c (+0.16) 1.34ab (+0.19) 116a (+0.86) 0 100 (F]N 2) 44.0a (+10.85) 0.08a (+0.02) 31.6a (+5.70) 0.06a (+0.01) 1.79c (+0.29) 163c (+1.90) 0.05 100 (F 2N 2) 52.7a (+6.25) 0.12a (+0.01) 45.3a (+3.46) 0.10a (+0.02) 2.27d (+0.31) 161c (+0.99) 5.0 100 (F 3N 2) 146.3b (+28.96) 0.36b (+0.09) 147.0b (+26.19) 0.36b (+0.08) 2.45d (+0.29) 177d (+3.70) In each column, means with a different suffix are significantly different at the 5-percent level. Numbers i n parenthesis are standard deviations. 4 8 TABLE 12. Analysis of variance of fo l i a r nutrient composition with varying supply levels of iron and nitrogen. Source Degrees of of N P K Ca Mg S Al B Cu AFe Fe Mn Zn variation freedom Iron 2 Nitrogen 1 Iron x 2 Nitrogen Error 42 Total 47 ns * ns ** ** AA AA AA AA AA ** ns ns ** ns AA A A A A A A n s AA n s ns ** ns ns A A A A A A A A AA A A A A A A A A A A u g A A A A A > ns = Significant at the 0.05 and 0.01 levels, respectively. = Not significant. 2.0 H MASS OF 100 NEEDLES ( g ) i.oi 200 n I50-| SEEDLING HEIGHT ( m m ) 100 H 50 200 CONC. ( p p m ) N 2 0.25 CONTENT ( m FOLIAR TOTAL IRON F i g u r e 13 . R e l a t i o n s h i p between f o l i a r t o t a l i r o n c o m p o s i t i o n and s e e d l i n g growth ( l o d g e p o l e p i n e ) . 50 2.0 H MASS OF 100 NEEDLES (g) 1.0 0 2 0 0 150 SEEDLING HEIGHT (mm) 1 0 0 5 0 2 0 0 4 0 0 0 CONC. (ppm) r 0 . 25 0.5 CONTENT (mg) FOLIAR ACTIVE IRON Figure 14. Relationship between f o l i a r active iron composition and seedling growth (lodgepole pine). 51 30 or more ppm active iron, while those which were chlorotic because of iron deficiency, had less than 30 ppm. D. Summary of Greenhouse Experiments The primary aim of the greenhouse experiments was to bracket the c r i t i c a l deficiency value or range of boron, copper and iron for lodge-pole pine through the use of f o l i a r analysis data and seedling growth response. While direct extrapolation of the findings to f i e l d situa-tions has to be approached with caution, the results obtained signify some important practical applications. The findings suggest that the c r i t i c a l ranges expressed as concen-tration (ppm) are 7 to 16 for boron, 2 to 3 for copper, with the lower values representing c r i t i c a l levels for these elements. The c r i t i c a l levels for iron are estimated at 44 and 32 ppm for total and active iron, respectively. Concentrations below the c r i t i c a l level may be associated with acute deficiency of that particular element. Values above the c r i t i c a l range imply optimum or near optimum growth. The levels at which toxicity symptoms would appear have not been reached i n this study. There was a reciprocal relationship between nitrogen supply and fo l i a r composition of boron, copper, active iron and total iron. The composition of these three micronutrients in the foliage decreased as fo l i a r nitrogen concentration was increased by increasing nitrogen supply. This has a significant practical implication in that nitrogen f e r t i l i z a t i o n might induce deficiencies of these micronutrients. 52 CHAPTER 4. FIELD EXPERIMENT A. Introduction It i s well known that foliage of plants can rapidly absorb plant nutrients. There are two main paths of entry for f o l i a r nutrients: the cuticle and the stomata. A detailed description of the mechanism of f o l i a r absorption has been described by a number of authors including Boynton (1954), van Overbeek (1956), Wittwer and Teubner (1959), Jyung and Wittwer (1965) and Franke (1967). Foliar application of f l u i d f e r t i l i z e r s has been used in agricul-ture for a long time and has been an accepted practice in supplying various micronutrients for many crops (Murphy and Walsh 1972; Traynor 1980). This method of applying f e r t i l i z e r s offers many theoretical advantages for forestry. These are mainly related to avoiding s o i l reactions which reduce the availability of applied nutrients to the trees (Knight 1978; Mengel and Kirkby 1982). Foliar sprays can also correct nutrient deficiencies faster or maintain optimum nutrition of a particular nutrient better than could be accomplished by s o i l applica-tion. Miller and Young (1976) also indicated that f o l i a r nutrient application has more f l e x i b i l i t y i n application timing, reduces the potential for nutrient leaching and water pollution, improves logistics of delivery and application, and allows more options in f e r t i l i z e r formulations. 53 The main limitation in the use of f o l i a r application is that i t can cause severe f o l i a r scorching. This method, therefore, has not been generally successful in applying macronutrients because of the d i f f i c u l t y in getting significant quantities of these nutrients into the plants without causing serious injury to the foliage (Engelstad and Russel 1975). The use of weak solutions of macronutrient f e r t i l i z e r s in forestry has produced discouraging results. For example, Paavelainen (1972) in Finland reported no remarkable growth response and no significant nitrogen uptake by Scots pine sprayed with 0.7 percent urea solution. In the southeastern United States, the application of diammonium phosphate solution, containing 1.4 percent nitrogen, to slash pine seedlings did not increase nutrient uptake or growth of seedlings (Schultz 1968). In another greenhouse experiment with slash pine, Eberhardt and Pritchett (1971) observed needle burning at 0.4 percent nitrogen concentration, and the severity increased as nitrogen supply was increased. The only encouraging result was reported by Miller and Young (1976) who found i t feasible to apply urea-ammonium solutions containing 32 percent nitrogen to Douglas-fir/western hemlock stands at rates up to 224 kg of the f e r t i l i z e r per hectare. They reported an increase of 106 percent i n volume growth relative to untreated trees over a four-year period. There is very l i t t l e published information on f i e l d experimentation involving the use of micronutrient f e r t i l i z e r s as f o l i a r sprays in forestry. One isolated example is in New Zealand where micronutrient sprays have been used to correct boron, copper, iron and zinc deficien-cies of radiata pine in the nursery (Knight 1978; Mead and Gadgil 1978). 54 Nutritional surveys by f o l i a r analysis of several forest stands in interior British Columbia by Ballard (1981) indicated presumptive evidence of nitrogen, copper and iron deficiencies. This chapter reports findings from preliminary f i e l d investigation on the use of urea, ferrous sulphate and copper sulphate solutions as f o l i a r sprays on lodgepole pine and Douglas-fir. (A Douglas-fir stand on calcareous s o i l was selected because no l o g i s t i c a l l y suitable lodgepole pine stand on such a s o i l had been found by the time this part of the study commenced). The main aims of the study were: 1. To assess tree growth response to f o l i a r application of f e r t i l i z e r s . 2. To determine f o l i a r nutrient responses to fo l i a r applied nutrients. 3. To evaluate the potential for f o l i a r f e r t i l i z a t i o n to relieve copper and iron deficiencies. B. Methods and Materials 1. Site Descriptions (a) Site Locations The experiment was conducted at five different sites in the Prince George and Kamloops Forest Regions in interior British Columbia (Figure 15). Two of the sites, Tsus Creek (53°45' N, 121°49' W, elevation 854 m) and Opatcho Lake (53°44' N, 122°18' W, elevation 900 m) are in Prince George Forest Region. The remaining three sites are at Marshall Lake (50°56' N, 122°34' W, elevation 1540 m), Shulaps Creek (50°57* N, 122°18' W, elevation 1235 m) and Pavilion Lake (50°52' N, 121°43*W, elevation 905 135* 130* 123' 120* Figure 15. Map showing location of the study sites. 56 m) in Kamloops Forest Region. For brevity, Tsus Creek, Opatcho Lake, Marshall Lake, Shulaps Creek and Pavilion Lake sites w i l l be referred to as Sites 1, 2, 3, 4 and 5, respectively, for the remainder of the text. The two forest regions are generally characterized by dry cold climates. Normal precipitation for the year and for the growing season recorded at the nearest weather stations for Prince George area are 628 mm and 300 mm respectively, and that for the Kamloops area are 440 mm and 198 mm. Frost-free period (30 years average, 1951-1980) for Sites 1, 2, 3, 4 and 5 are 85, 85, 131, 198 and 56 days, respectively. In the Prince George area the coldest month is January with a normal daily minimum temperature of -12.1°C. The warmest month is July with a normal daily maximum temperature of 22.0°C. The minimum and maximum daily temperature for the Kamloops sites are -13.8°C (January) and 21.6°C (July), respec-tively (Environment Canada 1982). (b) Stand Characteristics Sites 1, 2, 3 and 4 consisted of mainly young even-aged lodgepole pine of natural origin. In 1982, the estimated stand age (at stump) for Sites 1, 2, 3 and 4 were 23, 23, 33 and 13 years, respectively. Douglas-f i r is the only tree species at Site 5. The estimated age of this stand is about 100 years. The lodgepole pine sites, with the exception of Site 3, are severely overstocked. For example, stand densities average 50,000 and 122,000 stems/ha at Sites 1 and 2, respectively (Dickey 1981). Sites 3 and 5, on the hand, are c r i t i c a l l y understocked. Tree heights at the lodgepole pine sites ranged from 1.5 to 5 m and diameter (dbh) varied 57 from 16 to 64 mm. The Douglas-fir trees have heights and diameters ranging from 2.7 to 6.5 m, and 38 to 124 mm, respectively. (c) Soil Characteristics Soil profile descriptions according to the Canadian system (Canada Soil Survey Committee 1978) are given in Appendix C. Soils at Sites 1, 2, 3, 4 and 5 are classified as Orthic Humo-Ferric Podzol, eluviated Dystric Brunisol, Orthic Gray Luvisol, Brunisolic Gray Luvisol, and Orthic Regosol, respectively. Table 13 shows some chemical s o i l characteristics of the s o i l profile excavated at each of the sites. Available water storage capaci-ties for the five soils were calculated based on Clapp and Hornberger (1978), using f i e l d estimates of rooting depth, s o i l texture, and coarse fragments content (Table 14). The chemical and physical s o i l character-i s t i c s were used only for site characterization and are not discussed In detail in the text. 2. Experimental Design The experiment was carried out based on single-tree plot technique (Viro 1967) using completely randomized design with five replicate trees per treatment at each si t e . There are a total of 14 treatment combina-tions. The nature and number of treatments varied from one site to another (Table 15). Within each site, careful selection of test trees was done to ensure homogeneity in stand characteristics and freedom from any deformity. The distance between any two test trees was as far apart as TABLE 13. Some chemical characteristics of s o i l profiles at the study sites. pH Total C Total N Available P DTPA-extractable cations Site Horizon H20 % % ppm C/N Cu Fe Mn Zn LFH 4.0 19.85 0.82 62.8 24.2 1.44 327.0 711.0 32.0 1 Ae 4.2 1.32 0.02 38.0 22.0 0.40 76.6 8.0 1.0 Bf 5.0 0.29 0.02 36.0 14.5 0.26 4.6 7.0 0.4 BC 5.1 0.13 0.01 36.0 13.0 0.16 6.2 2.1 0.1 LFH 4.6 19.83 0.75 52.2 26.4 1.12 399.2 993.4 20.2 2 Ae 4.7 1.23 0.05 17.0 24.6 0.88 106.0 45.0 1.9 Bf 5.5 0.64 0.04 56.0 16.0 0.28 33.4 4.9 0.3 BC 5.6 0.11 0.01 6.0 11.0 0.22 18.6 11.8 o.o: LFH 4.9 18.90 0.75 53.0 25.2 0.48 167.6 281.8 15.4 3 Ae 5.7 0.79 0.03 90.0 26.3 0.22 37.0 22.6 0.5 Bt 5.7 0.48 0.02 9.0 24.0 1.56 41.4 25.4 0.3 BC 5.7 0.24 0.01 2.0 24.0 1.38 23.4 14.8 0.3 LF(H) 5.7 19.81 0.84 37.4 23.6 0.66 243.2 501.8 41.4 4 AE 5.9 . 0.67 0.02 46.0 33.5 0.62 51.6 26.9 0.6 Bm 5.6 0.62 0.02 300.0 31.0 0.42 59.6 8.1 0.5 Bt 5.3 0.47 0.02 15.0 23.5 1.14 60.6 8.4 0.3 5 LFH 6.5 19.68 0.73 11.4 27.0 0.82 22.0 70.0 74.8 C 7.5 3.05 0.18 2.0 16.9 1.00 9.4 7.9 1.4 59 TABLE 14. Some physical s o i l characteristics of the study sites. Coarse fragment Available water Site Rooting depth Soil texture content capacity 3 —3 (mm) (mm mm ' (mm) 1 330 sandy loam 0.05 37 2 550 sandy loam 0.05 52 3 400 sandy loam 0.05 42 4 800 loam 0.15 88 5 500 sandy loam 0.50 28 60 possible to avoid any treatment effect of one tree upon another. Trees were identified with plastic tags to eliminate any contamination from tagging. 3. F e r t i l i z e r Application The f e r t i l i z e r s used were copper sulphate, ferrous sulphate and urea solutions, applied singly and also in combination with each other at different levels of concentrations and frequency of applications. There was a total of 14 treatment combinations involving 250 trees. The main t r i a l was initiated in spring, 1981 with the f i r s t appli-cation made just as the current year needles emerged. Sites 2, 3 and 4 received copper, iron and nitrogen treatments, whereas only iron and nitrogen treatments were applied at Sites 1 and 5. There was no reappli-cation in 1982 for the main t r i a l . Table 15 outlines details of the f e r t i l i z e r application. Treatment numbers as designated i n Table 15 w i l l be used throughout the text. Iron and nitrogen treatments were repeated on different trees in 1982 at Sites 1, 2, 4 and 5 (Table 16). Preparation of the f e r t i l i z e r solutions was done at the site. This involved dissolving and diluting to the required dosage in water. Tap water from Vancouver was used to prepare the solutions for treatments at the Prince George sites, whereas for the Kamloops sites, water was taken from Shulaps Creek. Chemical analysis of the water from both sources is given in Table 17. A commercial household detergent (trade name 'Joy'), at a concentration of 0.5 percent by volume was added to the f e r t i l i z e r solution as a surfactant to improve solution contact with needle surfaces and thereby enhance absorption. TABLE 15. F e r t i l i z e r treatments for the main (1981) t r i a l . 61 Treat- Frequency Site ment Treatment Date of of No. ap p l i c a t i o n a p p l i c a t i o n 1 Control 1 5 4% FeS0 4 (Tsus Creek) 7 2% urea 11 4% FeS04 + 2% urea June 18, July 8 June 18, July 8 June 18, July 8 1 Control 0 0 2 1% CuS0 A June 18, July 8 2 3 0.2% CuSO^ July 28 1 4 0.1% CuS0 4 July 28 1 5 4% FeS0 4 June 18, Ju l y 8 2 2 6 2% FeSO^ July 28 1 (Opatcho Lake) 7 2% urea June 18, July 8 2 8 1% CuS0 4 + 2% urea June 18, Ju l y 8 2 9 0.1% CuSO^ + 2% urea July 28 1 10 0.2% CUSO4 + 2% urea July 28 1 11 4% FeSO^ + 2% urea June 18, July 8 2 12 1% CuS0 4 + 4% FeS04 + 2% urea June 18, July 8 2 13 0.1% CUSO4 + 2% FeS04 + 2% urea July 28 1 14 0.2% CUSO4 + FeS04 + 2% urea July 28 1 1 Control 0 0 2 1% CUSO4 June 12, Ju l y 30 2 3 5 4% FeS04 June 12, July 30 2 (Marshall Lake) 7 2% urea June 12, July 30 2 8 1% CUSO4 + 2% urea June 12, July 30 2 11 4% FeS04 + 2% urea June 12, July 30 2 1 Control 0 0 2 1% CUSO4 June 11, July 29 2 3 0.2% CUSO4 July 29 1 4 4 0.1% CuS0 4 July 29 1 (Shulpas Creek 5 4% FeS04 June 19, July 29 2 6 2% FeS0 4 July 29 1 7 2% urea June 11, July 29 2 8 1% CUSO4 + 2% urea June 11 1 11 4% FeS04 + 2% urea June 19 1 12 1% CUSO4 + 4% FeS04 + 2% urea June 19 1 5 1 Control 0 0 (Pa v i l i o n Lake) 5 4% FeS04 June 11. July 10 2 7 2% urea June 11, July 10 2 11 4% FeS04 + 2% urea June 11, July 10 2 62 TABLE 16. F e r t i l i z e r treatments for the repeat (1982) t r i a l . Site Treatment No. Date of application Frequency of application 1 Control 5 4% FeSO-4 7 2% urea 11 4% FeSC-4 + 2% urea 0 July 4, July 29 July 4, July 29 July 4, July 29 0 2 2 2 1 5 7 11 Control 4% FeSO-4 2% urea 4% FeS04 + 2% urea 0 July 4, July 29 July 4, July 29 July 4, July 29 0 2 2 2 1 Control 5 4% FeS0 4 7 2% urea 11 4% FeS04 +2% urea June 29, July 30 June 29, July 30 June 29 July 30 0 2 2 1 1 Control 5 4% FeS04 7 2% urea 11 4% FeS04 + 2% urea 0 June 28, July 30 June 28, July 30 June 28, July 30 0 2 2 2 TABLE 17. Chemical analysis of water used for preparing f e r t i l i z e r solution. Vancouver tap water* Element (Capilano system) Shulaps Creek (ppm) (ppm) Chromium <0.001 0.017 Cobalt - 0.001 Calcium 1.4 19.0 Copper <0.001 0.002 Iron 0.13 0.038 Magnesium 0.19 8.8 Sodium 0.45 0.2 Potassium 0.12 0.2 Chloride 0.54 0.3 * Greater Vancouver Regional District, Water District Data. 64 Each f e r t i l i z e r solution was applied with a backpack plastic sprayer. The entire crown of a l l lodgepole pine trees was sprayed; Douglas-fir crowns were sprayed up to a height of about 6.5 m (using a ladder). Spraying continued u n t i l dripping from the canopy began. For combined f e r t i l i z e r treatments, the following sequence of application was followed: ferrous sulphate, copper sulphate and urea. The amount of f e r t i l i z e r solution needed for each treatment of five trees was between 1.5 and 2 l i t e r s . 4. Field Sampling and Measurements Foliage samples from each tree of the main t r i a l were collected i n October, 1981. Both current and previous-year foliage were sampled. Samples were taken by cutting some shoots with a pole pruner from the sprayed zone, approximately around the upper one-third of the tree crown. Each sample was kept in a plastic bag, transported to the laboratory and stored in a deep cold-storage room at -10°C before further processing. Collection of second-year foliage samples of the main t r i a l and f i r s t -year samples of the repeat t r i a l was done in October, 1982. Tree height and diameter measurements were also taken at this time for description of stand characteristics. A s o i l pit was excavated at each site for profile description. Soil samples were collected for each horizon for chemical analyses. Results of s o i l chemical analysis were used only for site characteriza-tion; they were not s t a t i s t i c a l l y analysed. 65 5. Sample Preparation and Measurements Foliar samples collected in 1981 were separated into the current and previous-year's (1980) growth portions. Foliar sample preparation is similar to that for the greenhouse samples, and has already been described in Chapter 3. The same procedure was followed for samples collected in 1982 except five shoots from each sample tree were saved for shoot length measurement. The shoot length for each year's growth for the three consecutive years was measured to the nearest millimeter. In the main t r i a l , these three values represent pre-fertilization, and first-year and second-year post f e r t i l i z a t i o n shoot growth. For the repeat t r i a l , only the previous and current-year shoot length were taken. The shoot length measurement gave a data base of 3750 values for the entire experiment. Dry mass of 100 randomly selected needles was also measured for each sample collected i n 1982. This provided a measure of first-year post-fertilization biomass response for the repeat t r i a l . For the main t r i a l , (assuming a possible increase but no decrease i n number of needle primordia, as a result of treatments) i t provided an estimate of minimum second-year biomass response. A total of 604 f o l i a r samples was prepared for chemical analyses. 6. Chemical Analysis of Foliar Samples The f o l i a r samples were analysed for total N, P, K, Ca, Mg, A l , B, Cu, Fe, AFe, Mn and Zn. Total sulphur was done only on some samples to assess whether the trees were sulphur-deficient. The procedures for the analyses have been described in Chapter 3. 66 7. Soil Sample Preparation and Analysis Mineral s o i l and forest floor samples were air-dried at room tem-perature (22°C). The s o i l samples were then sieved through a #10 (2.0 mm) stainless steel sieve. Forest floor samples were ground in a Waring blender to pass a 20-mesh sieve. The samples were analysed for pH, total N, available P, organic C, available Cu, Fe, Mn and Zn. Soil pH was measured in water using a glass electrode pH meter. A 1:2 and 1:8 soil:water suspension was used for samples from the mineral and organic s o i l horizons, respectively. Total N determination was by Kjeldahl digestion procedure (Bremner 1965) and the digests were analysed for N colorimetrically on the Autoanalyzer. Available P was measured in Bray's No. 1 solution and organic C by the Walkley-Black method (Allison 1965). Copper, Fe, Mn and Zn were extracted with DTPA extracting solution and the f i l t r a t e s analysed by atomic absorption spectrophotometer (Lindsay and Norvell 1978). 8. Assessment of F e r t i l i z e r Response Several variables can be used to evaluate response to f e r t i l i z e r application. Some common examples include height, diameter, basal area, total volume, merchantable volume, biomass production and f o l i a r nutrient status. Needle weight and f o l i a r nutrient composition have been used by Timmer and Stone (1978), Morrow and Timmer (1981), and Weetman and Fournier (1982). Whichever variable is used, Armson (1974) stated that the basic reason for applying f e r t i l i z e r s i s to bring about a significant increase in net photosynthate production which is translated into a desired form 67 of growth. Armson also suggested that variables selected should be most se n s i t i v e and also most l i k e l y to give the inve s t i g a t o r the i n s i g h t i n t o what i s going on i n b i o l o g i c a l terms when f e r t i l i z e r s are applied. For these reasons, f e r t i l i z e r response i n t h i s study was evaluated i n two ways: as tree growth response and as f o l i a r nutrient response. Tree growth response was evaluated i n terms of shoot (twig) length and dry mass of 100 needles. It i s assumed that these two parameters could be used as predictors of subsequent volume response. Weetman and Fournier (1982) used needle weight and f o l i a r nutrients as response v a r i a b l e s . For f o l i a r nutrient response, the elemental concentrations were expressed as percent f o r macronutrients, and ppm (= mg/kg), f o r micro-nutrients, both on an oven-dry bas i s . Twelve elements were analysed but emphasis i n the text i s on elements that were applied (copper, i r o n and nitrogen). (a) Evaluation of Shoot Length and F o l i a r Mass Response Shoot growth response to f e r t i l i z e r treatments was expressed as an average of r a t i o s between 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 lengths. To compare differences i n response among treatments the shoot length growth was calculated with the following formula, and expressed as percentage. [av(Af/Bf) - av(Ac/Be)]100 where: av = the average f o r a l l r e p l i c a t e s 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 68 f = f e r t i l i z e d c = control (unfertilized) The ratio Af/Bf i s an index of f e r t i l i z e r response as well as climatic (environmental) effects, whereas Ac/Be is an index of only climatic (environmental) influence. Hence the above formula provides an index of f e r t i l i z e r response alone. As indicated earlier, f o l i a r mass measurement was taken only in 1982. This gives an estimate of second-year f o l i a r mass response for the main t r i a l and current-year response for the repeat t r i a l . Comparison among treatments was calculated and expressed as a percentage with the formula: mf - mc mc where: m = foliage mass f = after f e r t i l i z a t i o n c = control (unfertilized). The above formula eliminates climatic (environmental) influence and permits expression of f e r t i l i z e r response only. (b) Evaluation of Foliar Nutrient Response The following formula was used to compare f o l i a r nutrient response among treatments within the same year: ( n f " n c ) 100 v nc where: n = nutrient element 69 f = after f e r t i l i z a t i o n c = control (unfertilized). The calculation of year-to-year variation of f o l i a r nutrient response for the same treatment was based on the formula: nf„ nc„ {-f- -) 100 nf^ nc^ where: n = nutrient element f 2 = second year after f e r t i l i z a t i o n fl = f i r s t year after f e r t i l i z a t i o n cl> c2 = f i r s t and second year control, respectively, (c) Foliar Nutrient Status Interpretation For interpretation, concentrations and some ratios of f o l i a r nutrients evaluated in this study were compared to those reported in the literature. In some cases, especially with copper, active iron and boron, results obtained from the greenhouse experiment were also used for the assessment. 9. St a t i s t i c a l Analysis The data for a l l parameters were subjected to a multi-way analysis of variance. The UBC Genlin program (Greig and Bjerring 1980) was used to perform this test. This program was found to be suitable for perform-ing tests in an unbalanced analysis of variance. The program is based on analysis of variance for unequal subclass numbers using the least-squares method (Steel and Torrie 1960). 70 Where effects of main factors (treatment, year and site) were significant, the Duncan New Multiple Range test was performed for mean separation and to evaluate the order of magnitude of the difference. In situations where f i r s t - and second-order interactions were significant, variance components were partitioned and trend analysis was applied ( L i t t l e 1981). This involved descriptive interpretation of the results based on graphical data representation. Calculations were done using equations described in section 8 of this chapter. C. Results and Discussion In the following sections, effects of the main factors (treatment, year and site) as evident from f i e l d observations, and results from laboratory analysis and measurements w i l l be presented. Shoot growth response for the main t r i a l includes current (1981) and second-year (1982) assessment, and for f o l i a r mass i t is only a second-year response evaluation. Foliar analysis results for the main t r i a l represent previous-year (1980), current-year (1981), and second-year (1982) responses. In the repeat t r i a l , only current-year (1982) measurement i s presented for shoot growth and f o l i a r mass production. Foliar analysis results represent previous-year (1981) and current-year (1982) response. It must be remembered that the samples of previous-year foliage of both main and repeat t r i a l s also received f e r t i l i z e r treatments; no sample collection was done prior to f e r t i l i z a t i o n . 71 1. Foliar Scorching (a) Copper Treatments Among the 14 different combinations of treatments (Table 15), those involving the use of 1 percent copper sulphate solution applied alone or in combination with ferrous sulphate and/or urea caused very severe scorching and burning of the needles (Appendices D.l, D.2, D.3). This was observed at a l l the three sites (Sites 2, 3 and 4) receiving the above treatments. The observation was made six weeks after the f i r s t application, but presumably the injury happened quite soon after treat-ments were applied since f o l i a r absorption of nutrient elements i s a rapid process (Boynton 1954; Wittwer and Tuebner 1959). The scorching is probably due to copper sulphate. Apparently even a single application of 0.2 percent copper sulphate solution (5000 ppm Cu) i s toxic for lodgepole pine. Oldenkamp and Smilde (1966) made no mention of any toxic effect when two applications of 1 percent copper sulphate solution were made to eight-year old Douglas-fir. In the nursery, concentrations of 0.05 to 0.125 percent copper sulphate spray are common rates of application (Stone 1968). Lyle (1972) recommended a higher rate of 0.8 percent copper sulphate spray for loblolly pine seedlings in the nursery. It is obvious that different tree species differ in their tolerance to copper. However, at the end of the f i r s t growing season following f e r t i l i -zation in 1981, new and healthier foliage was produced on these treated trees (Appendices D.l, D.2, D.3). There was increased vegetative growth in 1982, the second year after f e r t i l i z a t i o n (Appendices D.4, D.5, D.6). 72 For both years, the f o l i a r colour was much greener than the foliage of the control tree (Appendix D.7). (b) Iron Treatments There was minimal scorching of needles following the f i r s t applica-tion of 4 percent ferrous sulphate alone or in combination with 2 percent urea on lodgepole pine (Appendices D.8, D.9). Needle burn was slightly more serious for ferrous sulphate plus urea treatment than ferrous sulphate alone, but less severe than treatments that included copper sulphate (discussed i n previous section). Recovery from the injury during the current-year growing season was better than for copper-treated trees but increased vegetative growth took place during the 1982 growing season (Appendices D.10, D . l l ) . There was no apparent f o l i a r injury with 2 percent ferrous sulphate treatment. This would seem to be a suitable dosage for lodgepole pine. For Douglas-fir, 4 percent ferrous sulphate applied alone or with urea did not cause any visible f o l i a r damage (Appendices D.12, D.13). In this study, Douglas-fir apparently could tolerate a higher level of iron in the foliage than lodgepole pine. In contrast, Korstian et a l . (1921) found that two sprayings of 2 percent ferrous sulphate on western yellow pine and Douglas-fir seedlings caused very severe injury to the needles. Lyle (1972) recommended a spray solution of 2.8 percent ferrous sulphate to correct iron deficiency i n the nursery. (c) Nitrogen Treatment There was no evident f o l i a r injury from repeated applications of 2 percent urea for both lodgepole pine and Douglas-fir (Appendices D.14, D.15). This dosage of two applications per growing season therefore seems to be a safe dosage for these two species. Miller and Young (1976) applied 32 percent nitrogen as urea-ammonium solution to Douglas-fir/ western hemlock stand and experienced serious scorching of the needles. However, they indicated that burning of up to about 30 percent of the needle surface is acceptable. (d) Causes of Needle Burn It seems appropriate at this stage to explain the differences in severity of scorching observed among treatments, especially in the case of lodgepole pine. As mentioned earlier, the most severe needle burn resulted from combined application of copper sulphate, ferrous sulphate and urea; moderate burn from ferrous sulphate with urea; minimal burn from ferrous sulphate alone, and no burning from urea alone. Needle burning caused by f o l i a r treatments of nutrient solutions has been referred to as "osmotic burning" by Miller and Young (1976). It i s a c e l l rupture due to large difference i n osmotic pressure across the c e l l wall due to the concentrated f e r t i l i z e r solution outside the c e l l . To equalize the pressure, f l u i d tends to move out of the c e l l and the f e r t i l i z e r solute moves i n . If this transport process cannot occur fast enough, due to large amounts of solute moving in, the c e l l ruptures (Miller and Young, 1976). It is probable that copper sulphate and ferrous sulphate solutions were too concentrated and this caused a build-up of pressure outside the c e l l . 74 Furthermore, the sulphate forms of f e r t i l i z e r s (which were used i n this study) are considered as low-analysis f e r t i l i z e r s which have high salt index (Tisdale and Nelson 1975). High salt index f e r t i l i z e r s have a higher tendency to cause osmotic burning. Another related possible reason why copper sulphate and/or ferrous sulphate caused higher Injury when used with urea i s that urea improves the permeability of the cuticle and thus favours diffusion of solutes (Franke 1967). This increased the pressure differential across the c e l l wall, and with high concentration of copper, iron and sulphate ions, led to c e l l rupture. "Osmotic burning" i s presumably not the only reason for the severe needle damage caused by copper sulphate. The concentration (mole fraction) of solute in 1 percent copper sulphate i s much lower than that in the much less injurious 4 percent ferrous sulphate solution. Thus i t appears that copper toxicity occurred where solute concentration was quite tolerable. In fact, copper sulphate has been used as an effective herbicide (Reuther and Labanauskas 1966). The mechanism of micronutrient toxicity is not well understood, but according to Kramer and Kozlowski (1979), toxicity of these elements, including copper, is mainly related to their injurious effects on enzyme systems. The addition of detergent as a surfactant was to enable the spray solution to form a film over the leaf surface, Instead of forming drop-lets . The principal advantage of this i s enhanced nutrient uptake by the leaf. A second advantage, according to Neumann and Prinz (1975) is that burn damage may be reduced. However, i t was noticed in this thesis research that the spray solution formed droplets hanging from the needle 75 surfaces. This led to localization of the f e r t i l i z e r solution and might have aggravated f o l i a r injury. 2. Tree Growth Responses (a) Shoot Growth (i) Lodgepole pine There was no significant response to treatments in lodgepole pine shoot growth during the current-growing season for both the main and repeat t r i a l s (Table 18). Treatment-site interaction was also not signi-ficant. There was, however, a highly significant difference (P _< .01) among treatments and sites for the second-year shoot growth of the main t r i a l . Treatment-site interaction was also highly significant. Trend analysis w i l l therefore be employed to interpret treatment responses. Figures 16 to 19 ill u s t r a t e the relationship between shoot growth and treatments. In general, there was a definite relationship between shoot growth and fo l i a r burning discussed i n previous section. The highest response in shoot increment during the second-year of growth at a l l four sites was from treatments that caused l i t t l e or no f o l i a r burn, mainly treatments 4, 7 and 11 (Figures 17 to 19). There was an average of 49 percent increase for these treatments. Treatment 5, which also caused minimal f o l i a r burning, gave inconsistent response from one site to another. At Sites 1, 2 and 3 i t increased shoot length by 44, 4 and 42 percent, respectively, but gave no response at Site 4. Shoot incre-ment for treatments that caused severe needle burn in fact was lower than that of untreated trees. This effect was more pronounced from treatments 76 TABLE 18. Analysis of variance of tree growth response variables for lodgepole pine and Douglas-fir. Treatment Species Source of variation Treatment Site x site Lodgepole pine: Main t r i a l First-year shoot growth Second-year shoot growth Second-year foliage mass Repeat t r i a l First-year shoot growth First-year foliage mass Douglas-fir: Main t r i a l First-year shoot growth Second-year shoot growth Second-year foliage mass Repeat t r i a l First-year shoot growth First-year foliage mass *,** = Significant at the 0.05 and 0.01 levels, respectively, ns = Not significant. - = Not available (only one Douglas-fir s i t e ) . ns ** A* ** ns ns ** ns ns ns ns * ns ns ns ** ** ns ns T R E A T M E N T - - N o . - -FeSO 4 U r e a % 1 .5 7 11 4 4 2 2 1.6 i SHOOT GROWTH RATIO 1.2 -/ 0.8 1 1 r I 5 7 TREATMENT NUMBER Figure 16. Second-year shoot growth ratio i n relation treatments at Site 1 (lodgepole pine). 78 T R E A T M E N T C u S O u F e S O „ U r e a - - N o . % - - - - - - -1 . . . 2 1 3 0 . 1 4 0 . 2 5 4 6 2 7 2 8 1 - 2 9 0 . 1 - 2 1 0 0 . 2 - 2 11 . 4 2 12 1 4 2 1 3 0 . 1 2 2 14 0 . 2 2 2 1.5 1 SHOOT GROWTH RATIO 1.0 H / \ / \ / \ / \ v \ / \ i \ i w \ / \ / \ l \ I M \ / \ / \ / \/ \ \ 0 . 5 T 1 1 1 1 1 1 ' « 1 r 1 1 2 3 4 5 6 7 8 9 10 II 12 13 14 TREATMENT NUMBER Figure 17. Second-year shoot growth ratio in relation to treatments at Site 2 (lodgepole pine). 79 3.5n TREATMENT — No . 1 2 5 7 8 11 CuS0 \ F e S 0 \ U r e a 4 4 2 2 2 SHOOT GROWTH RATIO 2.5-N / 1.5 T I 2 5 7 8 II TREATMENT NUMBER Figure 18. Second-year shoot growth ratio in relation to treatments at Site 3 (lodgepole pine). 8 0 T R E A T M E N T C u S 0 4 FeSO , , U r e a - - N o . - - - % 1 -2 1 3 0 . 2 4 0 . 1 5 4 6 2 7 2 8 1 - ? 11 - 4 2 12 1 4 2 1.6 -SHOOT GROWTH RATIO 1.2 -\ \ \ i \ i \ i \ / \-/ V / \ • \ \ \ / \ 0.8 — i 1 1 1 1 1 1 1 1 » 1 2 3 4 5 6 7 8 II 12 TREATMENT NUMBER Figure 19. Second-year shoot growth ratio in relation to treatments at Site 4 (lodgepole pine). 81 2, 8 and 12 at Site 2 (Figure 17) and also treatment 2 at Site 3 (Figure 18). There was an average decrease of about 26 percent in shoot length from these treatments. Phytotoxicity effects could be the reason for the negative response. This could also be the reason for the non-significant or delayed response during the first-growing season following f e r t i l i z a t i o n . There was, however, a highly significant (P _< 0.01) first-year nutrient uptake (section 3 of this chapter), and one would expect a corresponding signi-ficant growth response, which did not take place. It might have been possible that certain physiological mechanisms involving the enzyme systems in the tree did not increase the tree's photosynthetic rate until the second year. This was also reflected by needle length increment at Site 1, which did not respond in the f i r s t year but responded greatly i n the second year (Ballard and Majid 1984). As for site differences, the highest response occurred at Site 3. As mentioned earlier, Site 3 i s sparsely populated compared to the severely overstocked stands at Sites 1, 2 and 4. Therefore, there was less competition for light, moisture and other growth requirements for trees at Site 3. ( i i ) Douglas-fir As in the case for lodgepole pine, there was no significant effect of treatments on Douglas-fir shoot growth during the first-year growing season, but there was a highly significant (P <^  .01) response in the second-growing season (Table 18). Figure 20 compares individual treat-ment means relative to shoot growth. Treatment 11 (a combination of TREATMENT F e S C \ U r e a No . - - % 1.5 i SHOOT GROWTH RATIO 1.0 H 0.5 Figure 20. I 5 7 TREATMENT NUMBER II Second-year shoot growth ratio in relation to treatments at Site 5 (Douglas-fir). (The value outside the range delineated by the vertical bar is significantly different, P < 0.05). 83 iron and nitrogen) produced the highest response, an increase of 44 percent i n shoot length compared to the 1980 pre-fertilization growth. In summary, the highest response in shoot growth took place during the second-year of growth, and from treatments that caused minimal f o l i a r burn. Among treatments tested, the combined iron and nitrogen applica-tion, and copper applied as 0.1 percent copper sulphate were most promising. (b) Foliar Mass (i) Lodgepole pine Analysis of vari ance revealed highly significant (P K. .01) d i f f e r -ence among treatments in f o l i a r mass production during the second-year of growth following f e r t i l i z a t i o n in the main t r i a l (Table 18). There was no significant treatment-site interaction. No significant first-year treatment response was detected in the repeat t r i a l . Figure 21 compares individual treatment means in relation to fo l i a r mass of lodgepole pine. Treatment 4 was significantly different from a l l other treatments (P <^  .05). It produced the highest positive response, an increase of 77 percent in needle mass relative to the untreated trees. A l l the other treatments also resulted in significant positive response, but were not significantly different from one another (P <^  .05). The average increase in foliar mass for these other treat-ments ranged from 34 to 61 percent. For the nine copper treatment combinations, the general trend seems to indicate that a lower copper dosage applied once per growing season (treatments 3, 4 and 13) resulted in higher f o l i a r mass production than 84 I .5 T R E A T M E N T - - N o . - -C u S C \ F e S O u U r e a 2.0T l 2 3 4 5 6 7 8 9 10 11 12 13 14 1 0 . 1 0 . 2 1 0 . 1 0 . 2 1 0 . 1 0 . 2 4 2 4 4 2 2 2 2 2 2 2 2 2 2 FOLIAGE MASS (g) \ \ 1.0 n 1 1— 12 13 14 -i 1 r 2 3 4 5 6 ~T~ 7 n r 8 9 ~T r 10 II TREATMENT NUMBER Figure 21. Foliar mass of lodgepole pine in the second growing season in relation to treatments. (Values outside the range delineated by the vertical bar are significantly different, P < 0.05). 85 higher copper dosage applied twice during the growing season, as in treatments 2 and 8. This might be due to phytotoxicity as discussed earlier. A different trend was observed in the case of iron treatments. Instead, a higher dosage (treatment 5) gave better response than lower iron application as in treatment 6. Presumably because f o l i a r damage was minimal for the higher iron dosage. Iron and nitrogen treatments each applied alone gave better response than when applied together as in treatment 11, but these were less effective than treatment 13 which combined a low copper dose. ( i i ) Douglas-fir There was also a highly significant difference (P <^  .01) among treatments for second-year Douglas-fir f o l i a r mass production in the main t r i a l (Table 18). Figure 22 shows the relationship between f o l i a r mass and treatments. Treatment 5 produced the highest response, an increase of 67 percent over that of untreated trees, followed by a 58 percent increase from treatment 7. Foliar mass from treatment 11 (which produced the highest response in shoot increment) was, however, not significantly different from that of untreated trees. There was no significant treat-ment effect on f o l i a r mass production in the repeat t r i a l (Table 18). T R E A T M E N T F e S C \ U r e a - - N o . - - % 0.5 1 FOLIAGE MASS (g) 0.4-0.3 -0 J — i 1 1 r ~ I 5 7 II TREATMENT NUMBER Figure 22. Foliar mass of Douglas-fir in the second growing season in relation to treatments. (Values outside the range delineated by the vertical bar are significantly different, P < 0.05). 87 3. Foliar Nutrient Responses Treatment, site, year and their interactions a l l had a highly significant effect (P <^  0.01) on f o l i a r nutrients and aluminium in the lodgepole pine main t r i a l (Table 19). The only exception is zinc. Table 20 shows the contribution of each factor in terms of percentage variance component. In the repeat t r i a l , treatments had a significant effect on most f o l i a r nutrients except potassium, magnesium, boron and zinc (Table 21). Treatment-site interaction was significant for aluminium, copper, total iron and active iron. Table 22 gives the percentage variance component for each factor. For the Douglas-fir main t r i a l (1981 treatments), Table 23 shows the s t a t i s t i c a l differences among treatments, year and treatment-year interaction in f o l i a r nutrients. Treatment-year interaction was highly significant only for total and active iron. Table 24 gives the relative contribution of each of the factors to the total source of variation for these two variables. In the repeat t r i a l , nitrogen, aluminium, copper, total iron and active iron were significantly affected by treatments (Table 25). In the following sections, emphasis is on elements that were applied: copper, iron and nitrogen. The main focus of the discussion is on the main t r i a l . The trends of current year f o l i a r nutrient response to treatments for the above nutrients were similar in the repeat t r i a l . (a) Copper Treatments that included copper sulphate resulted in substantial increase in f o l i a r copper in both the previous and current-year foliage 88 TABLE 19. Analysis of variance for f o l i a r nutrient responses of lodgepole pine (main t r i a l ) . Source of variation N P K Ca Mg Al B Cu Fe AFe Mn Zn Treatment ** ** ** ** ns ** ** ** ** ** ns ns Year ** ** ** ** ns ** ** ** ** ** ** * Site * ** ** ** ** ** ** ** ** ** ** ** Treatment x Year ** ** * ns ns ns ns ** ** ** ns * Treatment x Site * ** ** ** ** ** ** ** ** ** ** jjg Year x Site ns ** * ** ** ** ** ** ** ** ** ns Treatment x Year x Site ns ns ns ns ns * ns ** ** ** ns ns *, ** = Significant at the 5 and 1 percent levels, respectively, ns = Not significant. TABLE 20. Percentage variance components i n relation to f o l i a r nutrient responses for lodgepole pine (main t r i a l ) . Percentage variation * Source of variation N P K Ca Mg Al B Cu Fe AFe Mn Zn Treatment 10.82 21.43 7.69 7.84 1.20 1.90 3.17 14.99 36.90 40.28 1.40 4.50 Year 28.25 21.43 22.44 32.03 0.86 3.86 1.19 6.07 22.84 16.75 8.25 1.51 Site 0.95 7.14 2.56 6.54 49.33 63.39 45.21 4.18 2.29 1.56 45.47 2.99 Treatment x Year 14.30 7.14 5.45 2.61 0.93 1.37 2.91 12.98 22.03 25.18 1.50 10.91 Treatment x Site 3.20 7.14 6.09 3.59 4.00 1.84 3.76 13.04 2.34 2.07 3.02 2.10 Year x Site 0.54 1.79 1.92 1.96 2.67 3.92 5.69 5.96 1.75 1.08 3.45 1.52 Treatment x Year x Site 3.54 2.86 3.21 2.29 2.67 2.36 1.68 14.38 2.27 2.01 2.28 3.79 Residual 35.94 28.57 43.27 36.27 28.00 17.67 25.60 30.18 9.90 11.79 25.43 70.38 * Percent variance components were calculated for variables where interactions were significant. TABLE 21. Analysis of variance for f o l i a r nutrient responses of lodgepole pine (repeat t r i a l ) . Source of variation N P K Ca Mg Al B Cu Fe AFe Mn Zn Treatment Site AA AA ns ** ns ns ns ** ns AA AA AA ns AA AA AA AA A AA AA A Treatment x Site ns ns ns ns ns AA ns AA A AA ns ns A AA = Significant at the 5 and 1 percent levels, respectively. ns = Not significant, TABLE 22. Percentage variance components in relation to fo l i a r nutrient response for lodgepole pine (repeat t r i a l ) . Source of variation Al Percent variation* Cu Fe AFe Treatment 8.37 Site 68.92 Treatment x Site 8.30 Residual 14.41 25.54 17.42 32.09 24.93 77.65 2.65 5.43 14.27 70.46 7.14 8.62 13.78 * = Calculated only where main factors interaction were significant. 91 TABLE 23. Analysis of variance for f o l i a r nutrient responses of Douglas-fir (main t r i a l ) . Source of variation N P K Ca Mg Al B Cu Fe AFe Mn Zn Treatment ** ** ns ns ** ns * ns ** ** ns ** Site ** ** ** ** ns ** ** ** ** ** ns ** Treatment x year ns ns ns ns ns ns ns ns ** ** ns ns *, ** = Significant at the 5 and 1 percent levels, respectively, ns = Not significant. TABLE 24. Percentage variance components in relation to f o l i a r nutrient responses for Douglas-fir (main t r i a l ) . „ c . Percentage variation* Source of variation ° Fe AFe Treatment 69.83 64.95 Year 9.27 12.02 Treatment x year 9.66 10.94 Residual 11.42 12.47 * Calculated only where interactions were significant. 92 TABLE 25. Analysis of variance for f o l i a r nutrient responses of Douglas-fir (repeat t r i a l ) . Source of variation N P K Ca Mg Al B Cu Fe AFe Mn Zn Treatment ** ns ns ns ns * ns ** ** ** ns ns *, ** = Significant at the 5 and 1 percent, respectively, ns = Not significant. (Figures 23 to 25). This undoubtedly proves the efficiency of nutrient absorption of copper by leaves as indicated by Rukovac and Wittwer (1957). The amount of copper absorbed was mainly related to the level of copper applied, though treatment differences existed from one site to another. At Site 2, the highest f o l i a r copper level was attained from treatment 2, followed in decreasing order by treatments 8, 12, 10, 3, 14, 9, 4 and 13 (Figure 23). Copper absorption was higher from treatment 8 than treatment 2 at Site 3 (Figure 24). At Site 4, the trend in decreasing order was treatments 12, 8, 2, 3 and 4. The common feature, however, was that the application of 1 percent copper sulphate resulted in higher f o l i a r copper than the lower rates of 0.1 and 0.2 percent. At Site 2, for instance, the actual f o l i a r copper concentration of the current-year foliage from treatment 2 is 112 ppm compared to about 10 ppm 93 1980 1981 1982 TREATMENT -- No. --1 2 3 4 5 6 7 8 9 10 11 12 13 14 C u S 0 H F e S O u % -U r e a 1 0 . 1 0 . 2 1 0 .1 0 . 2 1 0 .1 0 . 2 4 2 4 4 2 2 2 2 2 2 2 2 2 2 50 -1 FOLIAR Cu CONC. (ppm) 25 H 112 l f l I I I I I I • I \ 95 t / 5 i A !\ \ / V 2 3 4 T 1 1 1 1 1 1 1 1 6 7 8 9 10 II 12 13 14 TREATMENT NUMBER Figure 23. Foliar copper concentration in relation to treatments at Site 2 (lodgepole pine). 1 9 8 2 7 8 11 2 1 - 2 4 2 20-| FOLIAR Cu CONC. (ppm) IOH 1/ \ \ / .•••.\\ / Ni—-ft ' \ ' \ ' \ / \ / \ ' \ ' t * 7 A * 1 / \ \ I / \ x 1 \ \ i/ \\ i A. W 4 *T" 2 5 7 T 8 - i II T R E A T M E N T NUMBER Figure 24. F o l i a r copper concentration i n r e l a t i o n to treatments at Site 3 (lodgepole pine). 95 1 9 8 0 1 9 8 1 1 9 8 2 T R E A T M E N T C u S 0 u F e SO , , U r e a — N o . % 1 . . . 2 1 3 0 . 2 4 0 . 1 5 4 6 2 7 2 R 1 - 2 11 4 2 12 1 4 2 20 n FOLIAR Cu CONC. (ppm) 10 A I f \ \ / \ 51.5 A I At--/ / / / T 1 1 1 r-1 2 3 4 5 8 -i 1 II 12 TREATMENT NUMBER Figure 25. Foliar copper concentration in relation to treatments at Site 4 (lodgepole pine). 96 for treatment 4. Appendix E.l gives the mean f o l i a r copper values for a l l treatments in the main t r i a l . Iron and nitrogen applied individually or in combination with each other had no marked effect on f o l i a r copper level.- This was consistent at a l l the five sites, that i s , for both lodgepole pine and Douglas-fir (Figures 23 to 27). The f o l i a r copper concentration ranged from 1.5 to 4.2 ppm (Appendix E . l ) . The control trees had a similar range of copper level. This suggests that there i s no obvious external (treatment) or physiological interaction of either iron or nitrogen on copper when nutrients are fed to the foliage. Such interaction, however, was shown in the greenhouse experiments where nutrients were absorbed through the root system. As for yearly variation, there was a sharp decline in copper level in the second-year foliage of copper-treated trees (Figures 23 to 25). The general trend shows that treatments which produced a higher increase in both the, previous and current-year foliage tend to cause a bigger decrease i n the second-year foliage. These include treatments 2, 3, 8 and 12. The lowest decrease was from treatments 4, 9 and 13. For instance, f o l i a r copper concentration for treatment 2 at Site 2 decreased from 112 ppm to 7 ppm (151 percent decrease) as compared to a decrease of 6 ppm (119 percent reduction) for treatment 4 (Figure 23). The actual f o l i a r copper concentration of copper-treated trees in the second- year foliage at the three sites ranged from about 4 to 8 ppm. There are two implications that could be made here on the behaviour of foliar-absorbed copper. F i r s t , even though i t is considered not readily mobile in the plant (Mengel and Kirkby 1982), results from this 1980 1981 1982 T R E A T M E N T - - N o . - -1 5 7 11 F e S O u U r e a 4 4 2 2 3.0-FOLIAR Cu CONC. (ppm) 2.0-• * / / / - A ' \ / \ / \ / \ / \ / \ / \ / \ / • 1.0 " I — 7 - i II 5 T R E A T M E N T NUMBER Figure 26. Foliar copper concentration i n relation to treatments at Site 1 (lodgepole pine). 1980 1981 1982 T R E A T M E N T - - N o . - -1 5 7 11 F e S O , 4 4 U r e a 2 2 4 . 0 - . FOLIAR Cu CONC. (ppm) 3 .0 -\ \ V / 2.0 J , , , , I 5 7 II TREATMENT NUMBER Figure 27. Foliar copper concentration in relation to treatments at Site 5 (Douglas-fir). 99 experiment indicate that i t can be translocated to the newly produced, current-year foliage following f e r t i l i z a t i o n . This is evident from the high copper concentration in the newly-formed foliage sampled from trees that suffered severe f o l i a r burning (Figures 23 to 25). Secondly, the effect of f o l i a r application of copper i s short-lived. Loneragan (1981) hypothesized that copper entering leaves i s bound by nitrogen compounds such as proteins which are retained against transport even during development of copper deficiency. As shown in Appendix E . l , the f o l i a r copper values in the second-year foliage are mostly within the deficiency range reported i n the literature and also with that determined i n the greenhouse experiment. It seems appropriate at this stage to estimate the c r i t i c a l and toxic levels of copper for lodgepole pine under actual f i e l d conditions. Before making this assessment, i t i s f i r s t necessary to establish that tree growth response was not due to sulphur in copper sulphate. An organic N/organic S mass ratio of 14.6 was determined for radiata pine (Kelly and Lambert 1972) and for Douglas-fir (Turner et a l . 1977). This value presumably also applies to lodgepole pine. Tree growth i s not sulphur-limited i f the N/S ratio i s below this value. If i t is near or above 14.6 there might be sulphur deficiency, and as such, growth response might be due to sulphur application. In the three copper-treated stands the N/S ratio ranged from 9.6 to 12.6. Therefore, any growth response should be attributable only to copper effects. As mentioned earlier, a l l copper treatments produced significant positive response in biomass production. Among treatments involving the use of copper sulphate alone, the highest 100-needle mass was 2.04 g, 100 obtained from treatment 4 (Figure 21). Ninety percent of this would then be a 100-needle mass of 1.84 g, which is approximately the mass obtained from treatment 3 (Figure 21). The corresponding 1982 f o l i a r copper concentration of trees receiving this treatment averaged 4.1 ppm (Appendix E . l ) . Rounding off this value, i t i s suggested that lodgepole pine growing under similar f i e l d situations with f o l i a r copper below 4 ppm be considered as copper deficient. This value is slightly greater than that determined from the greenhouse experiment (2 to 3 ppm). Even though Stone (1968) says that copper toxicity i s uncommon i n forest trees, high f o l i a r copper levels have been found in trees at several locations in British Columbia (Ballard, personal communication), presumably because of copper ore associated with the s o i l parent material. Also, copper toxicity can be a serious problem i n forest areas neighbouring mineral ore processing plants (Lozano and Morrison 1981). In this experiment, the level of copper toxicity could only be inferred from the reduction of current-year shoot growth. The corresponding f o l i a r copper concentrations at Sites 2, 3 and 4 (Figures 23 to 25), where treatments started to depress shoot growth, ranged from 10.2 to 28.9 ppm, with an average value of 16.6 ppm. Therefore, i t i s suggested that copper toxicity i n lodgepole pine may occur whenever f o l i a r copper concentration exceeds about 17 ppm. This i s f a i r l y consistent with those values reported by Reuther and Labanauskas (1966), van Lear and Smith (1972) and, Lozano and Morrison (1982). 101 (b) A c t i v e Iron ( i ) Lodgepole pine The r e l a t i o n s h i p between active i r o n concentration i n the f o l i a g e and treatments at the four lodgepole pine s i t e s i s i l l u s t r a t e d i n Figures 28 to 31. As i n the case f o r copper, f o l i a r a p p l i c a t i o n of ferrous sulphate proved to be an e f f i c i e n t means of r a p i d l y increasing the active i r o n l e v e l i n the f o l i a g e . Both previous and current-year iron-treated f o l i a g e had high amounts of ac t i v e i r o n . Iron absorption, however, d i f f e r e d among d i f f e r e n t i r o n treatment combinations. A pronounced and i n t e r e s t i n g difference i s that i r o n applied with copper and nitrogen (treatment 12) produced the highest a c t i v e i r o n l e v e l i n the current-year f o l i a g e compared to i r o n plus nitrogen (treatment 11) or i r o n applied alone as i n treatment 5 (Figures 29 and 31). These three treatments used the same l e v e l of ferrous s u l -phate (4 percent). Where copper was not applied, treatment 11 resulted i n higher absorption of i r o n than treatments 5 or 6 (Figures 28 to 31). This trend i s consistent at a l l the four s i t e s . For i r o n applied alone, as i n treatments 5 and 6, higher absorption occurred with treatment 5 (Figures 29 and 31). This might be due to higher dosage from treatment 5. However, high i r o n supply did not necessa r i l y r e s u l t i n higher f o l i a r i r o n . For instance, a c t i v e i r o n concentration from treatments 13 and 14 which used 2 percent ferrous sulphate with copper and nitrogen was higher than from treatment 5 (Figure 29). According to Franke (1967) urea improves the permeability of the c u t i c l e . The extent of urea penetration through the c u t i c l e exceeds that 102 1980 1981 1982 T R E A T M E N T — N O . 1 5 7 11 FeSO^ U r e a 4 4 3001 FOLIAR ACTIVE Fe CONC. (ppm) 2 0 0 100-£ / // il -V-I 5 7 II TREATMENT NUMBER Figure 28. Foliar active iron concentration i n relation to treatments at Site 1 (lodgepole pine). T R E A T M E N T - - N o . - -FeS0\ -- % --1980 1981 1982 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 0 .1 0.2 1 0.1 0.2 1 0.1 0.2 4 2 4 4 2 2 2 2 2 2 2 2 2 2 3 0 0 n FOLIAR ACTIVE Fe -CONC . (ppm) 150 -0 - 1 - i 1 — i 1 — i 1 — i 1 1 — i 1 1 — i — i I 2 3 4 5 6 7 8 9 10 II 12 13 14 T R E A T M E N T NUMBER Figure 29. Foliar active iron concentration in relation to treatments at Site 2 (lodgepole pine). 104 1980 1981 1982 T R E A T M E N T - - N o . - -1 2 5 7 8 11 CuSO^ FeSO^ U r e a 4 4 2 0 0 FOLIAR ACTIVE Fe -I CONC . (ppm) 100-'I \ ^ • • • • • I 2 5 7 8 TREATMENT NUMBER II Figure 30. Foliar active iron concentration in relation to treatments at Site 3 (lodgepole pine). 105 T R E A T M E N T C u S 0 4 F e S 0 4 U r e a N o . - - - - % 1 -2 1 1 9 8 0 3 0 . 2 4 0 . 1 1 9 8 1 5 6 1 9 8 2 7 R 1 11 12 1 4 2 2 2 4 ' 2 4 2 300-| FOLIAR ACTIVE Fe . CONC. (ppm) 2 0 0 1 0 0 A / \ / \ / \ / \ / \ 1 * x / / ;/ ! ii ••• "/ • • ^5t=H=.^ • T 3 7 4 5 6 TREATMENT NUMBER -r 8 T 1 II 12 Figure 31. F o l i a r active i r o n concentration i n r e l a t i o n treatments at Si t e 4 (lodgepole pine). 106 of ions by 10 to 20 fold and this increased permeability for urea also favours f o l i a r absorption of ions applied with i t . This might explain the higher level of active iron when ferrous sulphate was applied with urea. Active iron level in the current-year foliage showed a significant increase resulting from copper application as 1 percent copper sulphate (treatment 2) but decreased for both the lower copper dosage as in treat-ments 3 and 4 (Figures 29 and 31). There was a higher increase i n fo l i a r active iron from nitrogen applied either alone or together with copper. Average active iron concentrations from these two treatments were 44 and 34 ppm, respectively, compared to 24 ppm for the control trees (Appendix E.2). This indicates that there i s a positive interaction of nitrogen and nitrogen plus copper on active iron. This i s contrary to the findings from the greenhouse experiments where increasing nitrogen supply decreased the active iron concentration in the foliage. As for yearly variation, there was a significant drop in active iron concentration i n the second-year foliage (Figures 28 to 31). The actual active iron values of iron-treated trees at the four lodgepole pine sites ranged from 25 to 46 ppm. The sharp decline in iron level i s probably due to i t s mobility. Iron has been known to be fixed i n the leaves with l i t t l e or no translocation to the growing region (Boynton 1954, Bukovac and Wittwer 1957, Hsu et a l . , 1982). Trends of total iron concentration w i l l not be discussed in detail, as they are the same as the trend for active iron, described above. (Appendix E.3 gives actual total iron values for a l l treatments and sites for the three years of foliage age). 107 The estimation of c r i t i c a l threshold value for active iron under f i e l d situations i s also done by f i r s t ascertaining that responses were due to iron alone and not to sulphur in ferrous sulphate. The use of N/S ratio discussed i n previous section on copper also applies for iron. A similar range of N/S values was found for the four iron-treated lodgepole pine stands. Therefore, as far as treatments are concerned, tree growth response that occurred can be attributed solely to iron, and not to sulphur. Among the two treatments using only ferrous sulphate (treatments 5 and 6), treatment 6 at Site 2 produced the highest shoot growth response (Figure 17). Ninety percent of this maximum shoot growth would then be 1.22 which is approximately the response obtained in 1982 by both treat-ments 5 and 6 at Sites 2 and 4 (Figures 17 and 19). The corresponding fo l i a r active iron concentration ranged from 25.0 to 32.8 ppm (Appendix E.2). Averaging these values, one could estimate that the c r i t i c a l f o l i a r active iron concentration is about 29 ppm, which agrees with the value reported by Zech (1970) for Scots pine. This is slightly below the c r i t i c a l range for active iron (32 to 45 ppm) determined in the green-house experiment (Chapter 3). ( i i ) Douglas-fir Figure 32 illustrates the relationship between fo l i a r active iron and treatments for Douglas-fir. There was also a substantial increase i n active iron concentration in both the previous and current-year foliage as a result of iron treatments. As for yearly variation in active iron level, there was also a sub-stantial decline from the f i r s t to second-year growing seasons (Figure 108 T R E A T M E N T FeSO , , U r e a 1980 No. -- % 1981 1 5 4 1982 7 2 11 4 2 FOLIAR ACTIVE CONC. (ppm) i 1 1 1 I 5 7 II TREATMENT NUMBER Figure 32. Foliar active iron concentration i n relation to treatments at Site 5 (Douglas-fir). 109 32). However, the mean active iron concentration in the Douglas-fir second-year foliage for treatments 5 and 11 were 79.3 and 89.5 ppm, respectively, and that of the control trees was 31.3 ppm (Appendix E.2). As mentioned earlier, the corresponding values for iron-treated lodgepole pine ranged from 25.0 to 46.3 ppm. It appears thaf Douglas-fir needles were able to retain the applied iron longer than lodgepole pine. No satisfactory explanation could be given for the above d i f f e r -ences. It i s obvious that different species reacted differently to foliar-applied iron. The possibility that iron was rendered more ava i l -able to tree roots during the second-growing season has to be excluded since s o i l iron status i s very low (Table 13). It could have been possible that some of the iron in the 1981 foliage was translocated to the 1982 foliage. Even though iron i s considered not readily mobile within the plant (Mengel and Kirkby 1982), i t can be translocated in the form of iron citrate to the growing regions of the plant ( T i f f i n 1972, Brown 1978). (c) Nitrogen (i) Lodgepole pine There was a highly significant difference (P _< .01) among treat-ments involving the use of 2 percent urea in terms of f o l i a r nitrogen concentration (Table 19). In general, a l l treatments caused an increase in f o l i a r nitrogen concentration of both the previous and current-year foliage of the four lodgepole pine stands, but with higher increase in current-year foliage (Figures 33 to 36). The only exception was at Site 4 where treatments 5 and 8 caused a decrease in nitrogen concentration of 1 9 8 0 1981 1 9 8 2 TREATMENT - - No . - -1 5 7 11 F e S O , U r e a 4 4 1.6 n FOLIAR N CONC. (%) 1.2 -0.8 J 1 1 1 I 5 7 TREATMENT NUMBER Figure 3 3 . F o l i a r nitrogen concentration i n r e l a t i o n to treatments at Site 1 (lodgepole pine). I l l 1980 1981 1982 T R E A T M E N T CuSO, , F e S O H U r e a - - N o . % -1 -2 1 3 0.1 - , 4 0.2 5 4 6 2 7 2 8 1 - 2 9 0 . 1 - 2 10 0.2 - 2 11 - 4 2 12 1 4 2 13 0.1 2 2 14 0.2 2 2 0.5 - " - i 1 1 1 1 1 1 " 1 1 ' 1 1 ' I 2 3 4 5 6 7 8 9 10 II 12 13 14 T R E A T M E N T NUMBER Figure 34. Foliar nitrogen concentration i n relation to treatments at Site 2 (lodgepole pine). 112 1980 1981 1982 T R E A T M E N T — N o . 1 2 5 7 8 11 C u S 0 4 F e S O ^ U r e a % 2 2 TREATMENT NUMBER Figure 35. Foliar nitrogen concentration i n relation to treatments at Site 3 (lodgepole pine). 113 T R E A T M E N T CuSO, , F e S O \ U r e a 1980 — N o - % 1 9 8 1 i . . . 1982 3 0 . 2 -4 0 . 1 5 4 - . 6 2 7 2 8 1 - 2 11 4 2 12 1 4 2 1.5-1 F O L I A R N C O N C . 1.2 1 / ^ \ / ' \ • f \. • / / / / • • • / • • • • • 0.9 H -i 1 1 1 1 1 r 2 3 4 5 6 7 8 T R E A T M E N T N U M B E R 12 Figure .36. Foliar nitrogen concentration in relation to treatments at Site 4 (lodgepole pine). 114 the previous-year foliage (Figure 36). In relation to the untreated trees, the increase in the current-year foliage ranged from 9 to 60 percent of the control values. The trend indicates that the application of urea alone (treatment 7) was less effective in increasing f o l i a r nitrogen concentration com-pared to when i t was applied with copper and/or iron, as in treatments 8, 11 and 12. For instance, f o l i a r nitrogen concentration in current-year foliage for treatment 7 ranged from 1.2 to 1.4 percent, and that from treatment 11 ranged from 1.4 to 1.5 percent (Appendix E.4). In fact, treatment 7 resulted in the lowest f o l i a r nitrogen in the current-year foliage as compared to the other treatments (Figures 35 and 36). It appears that both copper and iron have a synergistic effect on f o l i a r uptake of nitrogen in lodgepole pine. There was a decline i n fo l i a r nitrogen concentration for a l l the treated trees in the second-year foliage (Figures 33 to 36). As in the case for copper and active iron, the highest decrease was from treatments that originally caused higher nitrogen absorption, that i s , treatments 8, 11 and 12. The second-year f o l i a r nitrogen concentration of a l l treated trees at the four sites averaged 1.1 percent. The average value for the current-year foliage was 1.3. Using the c r i t i c a l value of 1.2 percent suggested by Swan (1972) and Binns e_t a l . (1980) for lodgepole pine, i t can be concluded that urea application was effective in increasing f o l i a r nitrogen to above the deficiency level during the year of application, but not in the following year. This indicates that the effect of fo l i a r application of urea i s also short-lived. 115 ( i i ) Douglas-fir There were highly significant (P j< .01) treatment and year effects on f o l i a r nitrogen concentration i n Douglas-fir foliage (Table 23). Treatment-year interaction was not significant (P <^  .05). Table 26 compares individual treatments means as well as yearly means for each treatment. As for treatment differences, treatments 5, 7 and 11 significantly increased f o l i a r nitrogen concentration in the current year foliage. There was, however, no significant difference among these three treat-ments. As for yearly variation, there was an apparent increase in fol i a r nitrogen from the f i r s t to second-year foliage for treatments 5 and 11, and a decrease for treatment 7. However, accounting for variation in the control trees by using the formula in Section B.8 of this chapter there was an actual decrease in fo l i a r nitrogen for treatments 5, 7 and 11. TABLE 26. Treatment and year effects on f o l i a r nitrogen concentration (%) of Douglas-fir. Year/ Treatment 1980a 1981a l a 5b 7 b l i b 0.86 0.76 0.86 0.80 0.90 0.91 0.76 0.93 0.95 0.88 0.98 0.90 Each value i s a mean of five trees. In each column and row, values designated by the same letter are not significantly different at the 5-percent level. 116 Therefore, the effect of applying urea to the foliage also did not last beyond the current-growing season. Based on the f o l i a r c r i t i c a l value of 1.2 percent for Douglas-fir (Binns et a l . , 1980) and on the f o l i a r nitrogen range from 0.8 to 0.98 percent attained by urea application, i t could be concluded that f o l i a r application of 2 percent urea failed to increase f o l i a r nitrogen level of Douglas-fir to above the c r i t i c a l value for nitrogen deficiency. D. Summary of Field Experiment F e r t i l i z e r solutions of copper sulphate, ferrous sulphate and urea were applied at different rates and various combinations to the crowns of lodgepole pine and Douglas-fir at various sites. The primary objective was to determine the effects of f o l i a r application of f e r t i l i z e r s on the growth and f o l i a r nutrient status. The following results were obtained: 1. The application of 1 percent copper sulphate alone or in combina-tion with ferrous sulphate and urea caused very severe needle burn in lodgepole pine. The optimum copper dosage for this species was at 0.1 percent copper sulphate. Moderate needle burn was observed on lodgepole pine treated with 4 percent ferrous sulphate, but not on Douglas-fir. Nitrogen applied as 2 percent urea did not cause any f o l i a r injury for either species. 2. There was a highly significant positive response in shoot growth and biomass production in both species during the second-growing season. At one of the lodgepole sites, needle length responded greatly during the second-year of growth. No positive tree growth 117 response was detected during the year of f e r t i l i z e r application. Treatments that caused minimal f o l i a r scorching gave the best response. These were 0.1 percent copper sulphate, 4 percent ferrous sulphate plus 2 percent urea, and urea applied alone at 2 percent. 3. Foliar application of copper and/or iron was highly effective in alleviating deficiencies of these elements in lodgepole pine, and iron deficiency in Douglas-fir. Foliar application was similarly highly effective i n alleviating nitrogen deficiency in lodgepole pine, but not i n Douglas-fir. Combined applications of f e r t i l i z e r s were more effective than individual application. However, the effects were only temporary and did not last beyond the current growing season. Foliar composition of these three elements was either within or approaching the deficiency range in the second growing season. Douglas-fir, however, showed a tendency to be able to retain more iron than lodgepole pine in the second year after f e r t i l i z a t i o n . 4. It is suggested that the c r i t i c a l level for copper in lodgepole pine growing under f i e l d conditions be tentatively set at 4 ppm, and the c r i t i c a l level for active iron at about 29 ppm. No estima-tion of the threshold values of these nutrients was made for Douglas-fir. 5. Nitrogen and/or iron treatments did not seem to have any physio-logical interaction with copper. The application of urea alone and urea with copper sulphate, however, increased the active iron concentration i n the foliage. 118 CHAPTER 5: CONCLUSIONS Two complementary studies, one in the greenhouse with lodgepole pine seedlings and the other in the f i e l d with lodgepole pine and Douglas-fir, were conducted to investigate some aspects of boron, copper, and iron nutrition of these two species. The greenhouse experiments were primarily designed to determine the threshold values of boron, copper and iron i n lodgepole pine. The f i e l d investigation involved f o l i a r applica-tion of copper, iron and nitrogen to lodgepole pine and Douglas-fir: (i) to determine the c r i t i c a l values of copper and iron for lodgepole pine under f i e l d conditions, ( i i ) to evaluate the potential of f o l i a r f e r t i l i -zation in relieving deficiencies of copper and iron, and ( i i i ) to assess tree growth and f o l i a r nutrient responses to f o l i a r f e r t i l i z a t i o n . For lodgepole pine grown under greenhouse conditions, the fo l i a r concentration c r i t i c a l ranges are 7 to 16 ppm for boron, 2 to 3 ppm for copper; for active and total iron, c r i t i c a l levels are 32 and 44 ppm, respectively. Findings from the f i e l d experiments with lodgepole pine indicated a c r i t i c a l value of 4 ppm for copper and 29 ppm for active iron. The threshold values for copper and iron estimated for seedlings grown in the greenhouse are f a i r l y comparable to those determined for trees growing under actual f i e l d conditions. These findings support the hypothesis made by Swan (1972) that f o l i a r nutrient concentrations associated with best growth are largely independent of tree age. It would not be grossly inaccurate to state that the practical significance 1 1 9 of the results obtained would be useful to correctly diagnose deficien-cies of copper and iron in lodgepole pine growing under f i e l d situations. For boron, however, f i e l d experimentation is needed to verify the results of the greenhouse study. Another important greenhouse finding that might have significant practical implications relates to the fact that an increase in f o l i a r nitrogen, as a result of increasing nitrogen supply, decreases f o l i a r composition of boron, copper and iron. In many forest stands in British Columbia, nitrogen deficiency is frequently a serious problem, and nitrogen f e r t i l i z a t i o n i s one of the management tools often contemplated. Such a practice might induce deficiencies of these three micronutrients, which otherwise would have been adequate for tree growth. In the f i e l d experiment, f o l i a r application of copper and iron proved to be an efficient means of alleviating deficiencies of these two nutrient elements in lodgepole pine, and iron in Douglas-fir. Applica-tion of 2 percent urea also proved to be effective in relieving nitrogen deficiency in lodgepole pine, but not in Douglas-fir. Combined nutrient applications were more effective than individual nutrient application. This is highly desirable economically since i t would reduce the costs of applying several elements. The above effects were, however, only temporary and did not last beyond the year of application, except for Douglas-fir, which showed a tendency to be able to retain more of the applied iron than lodgepole pine. In the case of lodgepole pine, f o l i a r copper, iron and nitrogen concentrations were within or approaching the deficiency range during the 120 second growing season. This warrants repeated applications of at least once every growing season, which might not be economically attractive. As far as dosage of application i s concerned, the use of 0.1 per-cent copper sulphate for lodgepole pine seemed to be the safest level, with no f o l i a r injury observed. Iron applied as 4 percent ferrous sulphate caused moderate fo l i a r burning for lodgepole pine but not i n Douglas-fir. A level of 2 percent ferrous sulphate was found to be suitable and equally effective for lodgepole pine. Nitrogen applied as 2 percent urea did not cause any f o l i a r injury for both species. It seems appropriate to conduct further tests with higher levels of urea to deter-mine whether f o l i a r nitrogen response would last for a longer period of time. Tree growth response to f o l i a r f e r t i l i z a t i o n measured in terms of shoot increment and biomass production were significantly positive for both species only during the second-year of growth. The non-significant response during the year of f e r t i l i z e r application could be due to either f o l i a r injury or possibly because of physiological processes that delay growth response. For lodgepole pine, the highest response was from treatments that caused minimal or no f o l i a r burning. These are 0.1 percent copper sulphate, 2 percent urea and 4 percent ferrous sulphate plus 2 percent urea. Shoot growth response for Douglas-fir was highest from 4 percent ferrous sulphate plus 2 percent urea but foliage mass was affected most by 4 percent ferrous sulphate followed by 2 percent urea. A longer term experiment is necessary to indicate i f tree growth responses associated with fo l i a r f e r t i l i z i n g are beneficial over long as well as short periods of time. Despite the short-lived effect on f o l i a r 121 nutrient status, f o l i a r f e r t i l i z a t i o n may deserve further consideration for f e r t i l i z e r application to forested lands. Another important area of research i n fo l i a r f e r t i l i z a t i o n that warrants further investigation is the effect of increasing the dosage of urea on the f o l i a r composition of boron, copper and iron. As indicated earlier for the greenhouse experiments, where nutrients were absorbed via the root system, an increase in f o l i a r nitrogen was associated with a decrease in f o l i a r boron, copper and total and active forms of iron. Foliar feeding of 2 percent urea in the f i e l d experiment, on the contrary, did not appear to induce copper deficiency, and in fact, increased the iron level in the foliage. If such a trend holds true with increasing supply of urea, f o l i a r f e r t i l i z a t i o n may, in the future, become an important and promising forest management tool in the Pacific northwest where nitrogen deficiency i s often encountered. In situations where both nitrogen and iron deficiencies occur, the application of urea alone, by f o l i a r feeding, could possibly alleviate deficiencies of both elements. It may also be worthwhile to conduct f i e l d experimentation on the effects of s o i l application of nitrogen f e r t i l i z e r s on f o l i a r micro-nutrient status to test the hypothesis that nitrogen f e r t i l i z a t i o n tends to induce micronutrient deficiencies. 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Soil Sci. Plant Anal. 2:363-374. Wolf, B. 1974. Improvement s in the azomethine—H method for the deter-mination of boron. Commun. Soil Sci. Plant Anal. 5:39-44. Woods, R.W. 1983. Trace element problems induced by heavy nitrogen f e r t i l i z a t i o n of Pinus radiata in South Australia. Commun. Inst. For. Fenn. 116:178-182. Zech, W. 1970. Nadelanalytische Untersuchungen uber die Kalkchlorose der Waldkiefer (Pinus s i l v e s t r i s ) . z.f. Pflanzenernahr. u. Bodenk. 125:1-6. Zottl, H.W. 1973. Diagnosis of nutritional disturbance in forest stands. In: FAO/IUFRO Int. Symp. on Forest Fertilization Proc, Paris, pp. 75-96. 132 APPENDIX A.l MODIFIED PARKINSON AND ALLEN DIGESTION FOR PLANT TISSUE ANALYSIS 1. Weigh 1 g (to the nearest mg) subsample of ground plant tissue (oven dried at 70°C for 3 hours) and place in 100 mL digestion tube. 30-35 tubes per set could be prepared, with a reference sample and a blank in each set. 2. Add 5 mL of cone. H2SO4 (reagent grade) to each sample, and mix on a mechanical vibrator immediately. 3. Dispense 1 mL of Li2S0^ - H2O2 mixture (prepared by mixing 7.0 g Li2S04, 0.21 g selenium powder in 175 mL 30% H2O2) into each tube. Wait unt i l reaction (foaming and spattering) ceases before continuing. 4. Repeat step 3. 5. Heat the rack of tubes on the digestion block at 360°C. Use discon-tinuous heating to overcome i n i t i a l foaming; that i s , 20-40 seconds on block, cool for about 2 minutes, 40-50 seconds of heating and cool, 1-2 minutes on block and cool for 5-10 minutes. 6. Add another 1 mL LIS04 - H 20 2 mixture to each tube. Wait t i l l reaction ceases. 7. Repeat step 6. 8. Digest on block for 1 1/2 hours at 360°C. 9. After 1 1/2 hours, remove rack from block. Add 0.5 mL H2O2 to each tube, return rack to block and digest for another 30 minutes. 10. Repeat step 9. Total digestion time is 2 1/2 hours. 11. Remove rack from block and allow digests to cool (approximately 1 hour). Samples should be pale yellow to milky white in colour. 12. Add about 80 mL of demineralized water. Allow to cool to room temperature before making to f i n a l volume (100 mL) with demineralized water. 13. Cover tubes with parafilm or inert stopper, invert 3-4 times to mix, and pour contents into a labelled 125 mL plastic bottle. 14. The original digest solutions are analyzed by atomic absorption spectrophotometry for Fe, Mn, Zn, Cu and Al. Total N and P are analysed on the auto-analyzer. A 25x dilution of the original digest solution i s made up for analysis of Ca, Mg and K on the atomic absorption spectrophotometer. 133 APPENDIX A.2 NITRIC ACID DIGESTION FOR ANALYSIS OF COPPER AND IRON IN PLANT TISSUE 1. Weigh 0.7 g (to the nearest mg) subsample of ground sample (oven dried at 70°C for 3 hours) and place sample into digestion tube. A set of 30-35 tubes could be prepared for one run, each set having a reference sample and a blank. 2. Add 5 mL cone. HNO3 to sample, mix by swirling and add an addi-tional 5 mL of HNO3. 3. Cover tubes with glass marbles to prevent s p i l l i n g and heat on digestion block at 40°C for 1 hour. 4. Increase heat up to 140°C and continue heating for 2 hours, counting from time the block reaches 140°C 5. Remove tubes from block and allow to cool. 6. Add about 7 mL demineralized water to each tube and mix by swirling. Allow to cool. Pour sample into a 25 mL measuring cylinder. Rinse digestion tube with demineralized water and add rinsings to the cylinder. Make volume up to 25 mL with demineralized water. Cover cylinder with parafilm and mix content by inverting at least three times. Pour contents into a 60 mL plastic bottle. 7. Analyze the solution for copper and iron by atomic absorption spectrophotometer. 134 APPENDIX A.3 PROCEDURE FOR ACTIVE IRON DETERMINATION IN PLANT TISSUE 1. Weigh 0.20 g of ground sample (oven-dried at 70°C for 3 hours) into a 60 mL screw-capped plastic bottle. 2. Add 10 mL IN HCI (reagent grade) in demineralized water to each sample. Tightly cap the bottle to prevent leakage. (70 samples could be prepared in one run). 3. Shake the bottle horizontally for 24 hours on a reciprocating shaker at room temperature. Have a blank and reference samples for each set. 4. F i l t e r the extract through Whatman No. 41 f i l t e r paper and collect the f i l t r a t e in a plastic bottle (previously calibrated and marked at 25 mL) or in a 25 mL volumetric flask. 5. Add 10 mL IN HCI to the 60 mL sample bottle, shake briefly by hand, and wash through the same f i l t e r paper into the plastic bottle or volumetric flask. 6. Make to 25 mL volume with IN HCI. 7. Analyze for active iron on atomic absorption spectrophotometer. Analysis should be done within 48 hours. APPENDIX B.l FOLIAR ELEMENTAL CONCENTRATIONS IN THE BORON EXPERIMENT Elements Treatment N P K Ca Mg S A l B Cu Fe AFe Mn Zn % ppm BlNl 1.10 0.21 0.67 0.36 0.25 0.25 0.006 11.7 4.4 339 332 294 47 (+0.08) (+0.04) (+0.03) (+0.05) (+0.04) (+0.03) (+0.001) (+1.7) (+0.5) (+91) (+90) (+59) (+14.9) B2Nl 1.12 0.21 0.76 0.38 0.26 0.32 0.006 43.4 4.2 352 332 300 50 (+0.06) (+0.03) (+0.06) (+0.03) (+0.02) (+0.03) (+0.001) (+5.4) (+0.7) (+119) (+106) (+55) (+15) B3N1 1.03 0.21 0.82 0.37 0.26 0.32 0.007 107.3 4.2 442 424 245 39 (+0.1) (+0.02) (+0.08) (+0.03) (+0.02) (+0.04) (+0.003) (+7.7) (+0.8) (+178) (+162) (+33) (+5.2) BlN 2 2.00 0.22 0.86 0.25 0.16 0.27 0.003 7.3 5.1 218 207 107 27 (+0.1) (+0.02) (+0.05) (+0.02) (+0.008) (+0.02) (+0.0005) (+3.0) (+0.3) (+62) (+80) (+11) (+29) B 2N 2 2.01 0.20 0.97 0.29 0.20 0.33 0.004 15.5 4.6 165 136 75 16 (+0.1) (+0.01) (+0.06) (+0.01) (+0.01) (+0.01) (+0.0005) (+2.1) (+0.6) (+32) (+38) (+6) (+2.8) B 3N 2 1.93 0.19 1.00 0.26 0.20 0.34 0.003 85.5 4.3 205 171 68 14 (+0.07) (+0.02) (+0.1) (+0.03) (+0.01) (+0.04) (+0.001) (+9.8) (+0.8) (+33) (+25) (+6.4) (+2.0) Each value i s the mean of 8 samples. The number i n parenthesis i s the standard deviation. APPENDIX B.2 FOLIAR ELEMENTAL CONCENTRATIONS IN THE COPPER EXPERIMENT Elements Treatment N P K Ca V _ Mg S Al B Cu Fe AFe Mn Zn ppm ClNl 1.11 0.21 0.85 0.31 0.22 0.28 0.006 103.9 1.1 349 323 198 43 (+0.08) (+0.02) (+0.08) (+0.04) (+0.02) (+0.02) (+0.002) (+10.8) (+0.5) (+130) (+126) (+29) (+11) C 2N! 1.10 0.20 0.84 0.39 0.27 0.34 0.007 111.7 2.5 349 297 309 50 (+0.07) (+0.02) (+0.08) (+0.06) (+0.03) (+0.04) (+0.003) (+7.3) (+1.1) (+66) (+45) (+52) (+12) C3N1 1.05 0.19 0.75 0.40 0.26 0.33 0.006 109.2 5.6 190 174 280 41 (+0.1) (+0.01) (+0.07) (+0.06) (+0.02) (+0.06) (+0.003) (+11.7) (+1.7) (+129) (+111) (+55) (+6.5) Cl«2 2.23 0.29 1.1 0.27 0.20 0.36 0.005 77.0 0.9 304 289 102 24 (+0.1) (+0.04) (+0.01) (+0.01) (+0.02) (+0.04) (+0.002) (+5.3) (+0.6) (+76) (+81) (+21) (+4.6) C 2N 2 1.97 0.21 1.1 0.27 0.19 0.34 0.003 77.1 1.8 160 139 68 16 (+0.1) (+0.02) (+0.01) (+0.01) (+0.02) (+0.03) (+0.001) (+5.9) (+1.6) (+37) (+29) (+7.8) (+3.1) C 3N 2 1.83 0.18 0.9 0.30 0.22 0.37 0.003 83.6 3.0 162 132 69 14 (+0.07) (+0.01) (+0.05) (+0.02) (+0.01) (+0.02) (+0.001) (+8.7) (+0.6) (+52) (+31) (+7.8) (+1.5) Each value i s the mean of 8 samples. The number i n parenthesis i s the standard deviation. APPENDIX B.3 FOLIAR ELEMENTAL CONCENTRATIONS IN THE IRON EXPERIMENT Elements Treatment N P K Ca Mg S A l B Cu Fe AFe Mn Zn F1N1 1.14 0.20 0.83 0.39 0.30 0.37 0.007 100.0 4.7 48.2 37.7 374 86 (+0.1) (+0.02) (+0.07) (+0.04) (+0.02) (+0.04) (+0.001) (+7.3) (+1.2) (+6.5) (+13.2) (+53) (+11.9) F 2Ni 0.98 0.20 0.79 0.36 0.28 0.29 0.006 97.2 3.2 81.4 54.8 338 71 (+0.08) (+0.02) (+0.06) (+0.04) (+0.03) (+0.03) (+0.001) (+12.8) (+0.4) (+30.3) (+14.3) (+87) (+17.5) F3Nl 1.09 0.18 0.75 0.43 0.27 0.35 0.008 103.5 3.7 359.4 351.1 287 41 (+0.09) (+0.01) (+0.08) (+0.05) (+0.03) (+0.05) (+0.001) (+8.2) (+0.5) (+127) (+120) (+39) (+6.3) F1 N2 1.74 0.19 0.90 0.36 0.26 0.40 0.007 99.1 3.9 44.0 31.6 136 24 (+0.1) (+0.02) (+0.06) (+0.04) (+0.02) (+0.04) (+0.002) (+8.5) (+0.9) (+10.9) (+5.7) (+26) (+6.7) F 2N 2 1.81 0.18 0.94 0.31 0.22 0.36 0.006 84.6 3.4 52.7 45.3 83 15 (+0.08) (+0.02) (+0.09) (+0.02) (+0.01) (+0.04) (+0.001) (+6.4) (+0.3) (+6.2) (+3.5) (+6) (+2.8) F3N2 1.84 0.18 0.90 0.30 0.22 0.36 0.006 89.6 3.5 146.3 147.0 66 13 (+0.06) (+0.01) (+0.09) (+0.01) (+0.01) (+0.03) (+0.001) (+5.5) (+0.6) (+28.9) (+26.3) (+7.6) (+1.6) Each value i s the mean of 8 samples. The number i n parenthesis i s the standard deviation. 138 APPENDIX C l SOIL PROFILE DESCRIPTION OF SITE 1 Horizon Depth (cm) Description LFH 5-0 Very dark gray (7.5 YR 3/0, moist); fresh and partially decomposed organic matter, fibrous, few, fine and medium roots; abrupt, smooth boundary; 4-6 cm thick; pH 4.0 (1/8 soil/H 20). Ae 0-2 Pinkish gray (7.5 YR 6/2, moist); sandy loam; single grain; loose, friable; slightly sticky, non-plastic; few, fine and medium horizontal roots; very few, fine, vesicular pores; abrupt, wavy boundary; 0-4 cm thick; about 5% coarse fragments; pH 4.2 (1/2 soil/H 20). Bf 2-12 Reddish yellow (5 YR 4/6, moist); loam; weak, fine to medium subangular blocky; friable, slightly sticky, non-plastic; few, fine, and plentiful medium horizon-t a l roots; very few, medium, oblique, matrix, vesicu-lar pores; clear, smooth boundary; 8-11 cm thick; about 5% coarse fragments; pH 5.0 (1/2 soil/H 20). BC 12-33 Brownish yellow (10 YR 4/6, moist); loamy sand, single grain; loose, friable; non-sticky, plastic; few, fine and plentiful, medium horizontal roots; very few, fine oblique, matrix, vesicular pores; gradual smooth boundary; 8-24 cm thick; about 7% coarse fragments; pH 5.1 (1/2 soil/H 20). C 33+ Dark yellowish brown (10 YR 3/6, moist); sand, single grain; loose, friable; non-sticky, non-plastic; few, fine, oblique, matrix, vesicular pores; about 15% coarse fragments, pH 5.6 (1/2 soil/H 20). CLASSIFICATION: ORTHIC HUMO-FERRIC P0DZ0L 139 Appendix C.l (Cont'd). A s o i l profile of Site 1 . 140 APPENDIX C.2 SOIL PROFILE DESCRIPTION OF SITE 2 Horizon Depth (cm) Description LFH 5-0 Very dark gray (7.5 YR 3/0, moist); semidecomposed organic matter; fibrous, abundant fine roots; abrupt, smooth boundary; 3-6 cm thick; pH 4.6 (1/8 S 0 H/H2O). Ae 0-6 Brown (7.5 YR 5/2, moist); loam, weak, medium, sub-angular blocky; friable, slightly sticky, non-plastic; abundant fine horizontal roots; few, fine, oblique vesicular pores; abrupt, wavy boundary; 4-7 cm thick; about 5% coarse fragments; pH 4.7 (1/2 soil/H 20). Bf 6-12 Brown to dark brown (5 YR 4/4, moist); sandy loam; weak, fine to medium subangular blocky; friable, slightly sticky, non-plastic; plentiful fine, horizontal roots; plentiful, medium, oblique, vesicular pores; clear wavy boundary; 5-8 cm thick; about 5% coarse fragments; pH 5.5 (1/2 s o i l / ^ O ) . BC 12-30 Strong brown (10 YR 5/6, moist); gravelly sandy loam; weak, fine to medium subangular blocky; friable, slightly sticky, non-plastic; few fine, horizontal roots; plentiful, medium, oblique, vesicular pores; clear, wavy boundary, 16-20 cm thick; about 25% coarse fragments; pH 5.6 (1/2 S 0 H/H2O). C 30-55+ Dark yellowish brown (10 YR 4/6, moist); gravelly sand, single grain; loose, friable; non-sticky, non-plastic; few, fine horizontal roots; few fine, oblique, vesicular pores; about 25% coarse fragments, pH 5.7 (1/2 soil/H 20). CLASSIFICATION: ELUVIATED DYSTRIC BRUNISOL Appendix C.2 (Cont'd). A s o i l p r o f i l e of S i t e 2. 142 APPENDIX C.3 Soil Profile Description of Site 3 Horizon Depth (cm) Description LFH 3-0 Very dark gray (7.5 YR 3/2, moist); fresh and partially decomposed organic matter; fibrous, few fine roots; abrupt boundary; 4-5 cm thick; pH 4.9 (1/8 soil/H 20). Ae 0-9 Light gray (10 YR 7/2, moist); very gravelly sand; (volcanic single grain; loose, friable, non-sticky, non-ash) plastic; few fine horizontal roots; gradual smooth boundary; 3-5 cm thick; about 40% coarse fragments; pH 5.7 (1/2 soil/H 20). Bt 9-14 Greyish brown (10 YR 5/2, moist); clay loam; moderate medium, subangular blocky; slightly hard, firm, sticky, slightly plastic; few fine and medium hori-zontal roots; very few, very fine, oblique vesicular pores; gradual wavy boundary; 8-10 cm thick; about 15% coarse fragments; pH 5.7 (1/2 soil/H 20). BC 14+ Brown (10 YR 5/3, moist); gravelly clay loam; weak to moderate, medium, subangular blocky; slightly hard, firm, slightly sticky, slightly plastic; few, fine and medium horizontal roots; few, fine, oblique, vesicular pores; about 25% coarse fragments; pH 5.7 (1/2 soil/H 20). CLASSIFICATION: ORTHIC GRAY LUVISOL 1 4 3 Appendix C.3 (Cont'd). A s o i l p r o f i l e of S i t e 3. 144 APPENDIX C.4 Soil Profile Description of Site 4 Horizon Depth (cm) Description LF(H) 3-0 Black (10 YR 2/1, moist); fresh and partially decomposed organic matter; clear, wavy boundary, 3-6 cm thick; pH 5.7 (1/8 soil/H 20). Ae 0-7 Light brownish gray (10 YR 5/2, moist); loamy sand; single grain; loose, friable, non-sticky, non-plastic; very fine and few, fine, horizontal roots; plentiful very fine, oblique, vesicular pores; clear wavy boundary; 6-8 cm thick; about 5% coarse frag-ments; pH 5.9 (1/2 soil/H 20). Bm 7-25 Brown (10 YR 5/2, moist); loamy sand, single grain; loose, friable, non-sticky, plentiful, very fine and few, fine horizontal roots; plentiful, very fine, oblique vesicular pores; clear, wavy boundary; 6-8 cm thick; about 5% coarse fragments; pH 5.6 (1/2 soil/H 20), Bt 25-80 Brown to dark brown (10 YR 4/3, moist); clay loam; moderate, medium, prismatic; slightly hard, firm, sticky, slightly plastic; few fine horizontal roots; very few, very fine, oblique, vesicular pores; clear, wavy boundary; 17-25 cm thick; about 15% coarse fragments; pH 5.3 (1/2 soil/H 20). CLASSIFICATION: BRUNISOLIC GRAY LUVISOL 145 Appendix C.4 (Cont'd). A s o i l p r o f i l e of S i t e 4. 146 APPENDIX C.5 Soil Profile Description of Site 5 Horizon Depth (cm) Description LFH 5-0 Dusky red (2.5 YR 3/2, moist); fresh and partially decomposed organic matter; abrupt, smooth boundary; 0-8 cm thick; pH 6.5 (1/8 soil/H 20). Ck 0-50+ Dark grayish brown (10 YR 4/2, moist); gravelly sandy loam; single grain; loose, friable, slightly sticky, non-plastic; few, very fine to fine horizontal roots; very few and fine, oblique, vesicular pores; about 50 % coarse fragments; pH 7.5 (1/2 soil/H 20). CLASSIFICATION: ORTHIC REGOSOL Appendix C.5 (Cont'd). A s o i l profile of Site 5. 148 Appendix D.2. Copper and nitrogen treatment: current-year growth (lodgepole pine). Appendix D.3. Copper, current i r o n and nitrogen treatment: -year growth (lodgepole pine). 151 Appendix D.4. Copper treatment: second-year growth (lodgepole pine). 152 Appendix D.5. Copper and n i t r o g e n treatment: second-year growth (lodgepole p i n e ) . 153 Appendix D .6. Copper, iron and nitrogen treatment: second-year growth (lodgepole pine). 155 Appendix D .8. Iron treatment: current-year growth (lodgepole pine). Appendix D.9. Iron and nitrogen treatment: current-year growth (lodgepole pine). 1 5 6 Appendix D.10. Iron treatment: second-year growth (lodgepole pine). 157 Appendix D . l l . Iron and nitrogen treatment: second-year growth (lodgepole pine). 158 Appendix D.13. Iron and nitrogen treatment: growth (Douglas-fir). current-year Appendix D.14. Nitrogen treatment: current year growth (lodgepole pine) Appendix D.15. Nitrogen treatment: current-year growth (Douglas-fir). APPENDIX E . l . MEAN VALUES FOR FOLIAR COPPER CONCENTRATION (PPM) OF LODGEPOLE PINE AND DOUGLAS-FIR IN RELATION TO TREATMENTS (MAIN TRIAL). Treatment 1980 1981 1982 Site 1 2 3 4 5 1 2 3 4 5 1 2 3 4 X 1.50 1.64 0.71 2.86 2.64 2.42 2.00 2.21 3.28 3.50 3.07 3.14 2.00 1.21 3.43 1. Control 0 0.30 0.48 0.00 1.69 0.41 0.53 0.60 0.58 0.64 0.69 0.32 0.30 0.78 0.41 0.20 n 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 X NA _ 19.02 9.05 NA NA 112.14 10.21 10.57 NA NA 6.50 5.14 3.65 NA 2. 1Z C11SO4 a NA - 6.50 6.08 NA NA 73.95 3.97 4.79 NA NA 1.17 2.43 1.05 NA n 0 0 4 3 0 0 3 5 5 0 0 5 5 5 0 X NA 19.11 NA 13.40 NA NA 28.93 NA 9.21 NA NA 3.86 NA 4.33 NA 3. 0.2Z C11SO4 a NA 1.58 NA 2.55 NA NA 16.14 NA 2.37 NA NA 0.53 NA 0.47 NA n 0 4 0 4 0 0 5 0 5 0 0 5 0 5 0 X NA 7.98 NA 6.25 NA NA 10.36 NA 5.86 NA NA 3.93 NA 3.86 NA 4. 0.1Z C11SO4 0 NA 2.58 NA 3.56 NA NA - 4.69 NA 2.31 NA NA 0.72 NA 0.59 NA n 0 3 0 4 0 0 5 0 5 0 0 5 0 5 0 X 1.50 2.22 3.29 1.07 2.79 3.14 3.71 2.21 3.21 3.93 3.43 2.71 2.86 4.00 4.00 5. 4Z FeS04 a 0.16 0.64 0.59 0.0 0.69 0.30 0.99 0.30 0.98 0.84 0.54 0.41 1.04 0.59 0.78 n 5 5 5 3 5 5 5 5 5 5 5 5 5 5 5 APPENDIX E . l . (cont'd) Treatment 1980 1981 1982 Site 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 X NA 1.97 NA 1.86 NA NA 2.71 NA 3.43 NA NA 3.36 NA 3.21 NA 6. 2% FeS04 a NA 0.20 NA 0.96 NA NA 0.41 NA 0.54 NA NA 1.15 NA 0.72 NA n 0 4 0 5 0 0 5 0 5 0 0 5 0 5 0 X 3.29 2.95 3.07 2.07 2.57 3.29 2.71 2.64 3.57 2.86 3.28 3.64 2.28 3.71 3.71 7. 2% urea 0 2.04 0.18 0.19 0.92 0.39 0.46 0.96 0.32 0.36 0.36 0.30 1.39 0.54 1.03 0.82 n 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 8. 1% CuS04 X NA _ 20.83 4.29 NA NA 95.09 12.93 11.64 NA NA 7.86 6.07 4.00 NA + 2% urea a NA - 10.00 0.0 NA . NA 87.0 4.83 3.14 NA NA 1.54 2.30 0.64 NA n 0 0 3 1 0 0 4 5 5 0 0 5 5 5 0 9. 0.1% CUSO4 X NA 13.21 NA NA NA NA 14.71 NA NA NA NA 4.50 NA NA NA + 2% urea a NA 0.0 NA NA NA NA 8.00 NA NA NA NA 1.67 NA NA NA n 0 2 0 0 0 0 0 0 0 0 0 5 0 0 0 LO. 0.2% CuS0 4 X NA 13.21 NA NA NA NA 33.22 NA NA NA NA 5.00 NA NA NA . + 2% urea a NA 0.0 NA NA NA NA 12.84 NA NA NA NA 1.43 NA NA NA n 0 1 0 0 0 0 5 0 0 0 0 4 0 0 0 APPENDIX E . l . (cont'd) Treatment 1980 1981 1982 Site 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 11. 4% FeS0 4 X 1.79 2.50 2.59 4.20 3.14 3.36 2.57 2.68 3.29 3.64 3.71 2.43 3.57 3.36 3.29 + 2% urea 0 0.0 0.62 0.18 1.00 0.30 0.78 0.93 1.14 0.46 0.64 0.60 0.16 0.80 0.82 0.59 n 1 3 4 3 5 5 5 5 5 5 5 5 5 5 5 12. 1% CuS0 4 X NA _ NA 51.50 NA NA 48.84 NA 15.79 NA NA 4.91 NA 5.71 NA + 4% FeS0 4 a NA - NA 0.0 NA NA 11.03 NA 8.94 NA NA 0.73 NA 1.52 NA + 2% urea n 0 0 0 1 0 0 4 0 5 0 0 4 0 5 0 13. 0.1% CuS0 4 X NA 4.82 NA NA NA NA 8.86 NA NA NA NA 4.07 NA NA NA + 2% FeS0 4 0 NA 0.25 NA NA NA NA 2.01 NA NA NA NA 1.30 NA NA NA + 2% urea n 0 2 0 0 0 0 5 0 0 0 0 5 0 0 0 14. 0.2% CuS0 4 X NA 4.29 NA NA NA NA 16.64 NA NA NA NA 3.64 NA NA NA + 2% FeS0 4 a NA 0.0 NA NA NA NA 11.25 NA NA NA NA 0.64 NA NA NA + 2% urea n 0 1 0 0 0 0 5 0 0 0 0 5 0 0 0 NA = Treatment not applied. - = Missing value. x = Average concentration value. a = Standard deviation, n = Number of samples. APPENDIX E.2. MEAN VALUES FOR FOLIAR ACTIVE IRON CONCENTRATION (PPM) OF LODGEPOLE PINE AND DOUGLAS-FIR IN RELATION TO TREATMENTS (MAIN TRIAL). Treatment 1980 1981 1982 Si t e 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 x 51.25 51.00 33.75 31.25 34.05 24.25 25.75 30.20 15.50 37.00 33.30 18.50 23.75 31.50 31.25 1. Control o 6.56 4.45 5.38 3.19 7.09 11.94 6.82 3.84 5.27 10.48 7.65 3.58 2.34 12.10 5.99 n 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 x NA - 35.31 70.42 NA NA 49.25 34.50 20.25 NA NA 20.00 22.25 38.50 NA 2. 1% CuS04 a HA - 4.83 46.07 NA NA 26.75 2.88 4.87 NA NA 5.30 6.02 8.90 NA n 0 0 4 3 0 0 3 5 5 0 0 5 5 5 0 x NA 35.00 NA 21.88 NA NA 23.25 NA 11.25 NA NA 20.30 NA 32.05 NA 3. 0.2% C11SO4 a NA 0.0 NA 6.65 NA NA 5.97 NA 2.65 NA NA 1.85 NA 5.62 NA n 0 4 0 4 0 0 5 0 5 0 0 5 0 5 0 x NA 40.42 NA 34.38 NA NA 25.50 NA 14.50 NA NA 26.00 NA 38.00 NA 4. 0.1Z C11SO4 0 NA 5.05 NA 16.85 NA NA 11.06 NA 5.77 NA NA 3.58 NA 6.94 NA n O 3 0 4 0 0 5 0 5 0 0 5 0 5 0 X 207.75 213.75 127.75 214.17 199.75 223.25 161.25 111.75 101.25 178.25 41.50 25.00 35.75 42.00 79.25 5. 4Z FeS04 a 34.62 58.18 26.08 63.84 61.83 105.14 23.78 16.24 31.46 36.79 3.79 6.25 9.30 9.42 13.39 n 5 5 5 3 5 5 5 5 5 5 5 5 5 5 5 APPENDIX E.2. (cont'd) Treatment 1980 1981 1982 Site 1 2 3 4 5 1 2 3 4 5 1 2 3 4 X NA 153.75 NA 78.75 NA NA 165.50 NA 76.50 NA NA 32.75 NA 30.75 NA 6. 2% F E S O 4 a NA 23.32 NA 33.92 NA NA 60.93 NA 40.46 NA NA 8.50 NA 11.10 NA n 0 4 0 5 0 0 5 0 5 0 0 5 0 5 0 X 38.00 24.06 27.81 30.00 40.25 26.00 35.05 38.50 29.25 23.76 36.00 26.00 23.00 29.75 30.! 7. 2% urea a 27.82 7.86 3.89 11.69 9.32 8.72 3.41 6.75 13.71 2.65 2.71 3.89 6.22 2.85 4.: n 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 8. 1% C U S O 4 X NA _ 28.33 34.25 NA NA 54.50 37.00 33.40 NA NA 25.75 30.75 27.75 NA + 2% urea a NA - 9.71 0.0 NA NA 15.58 2.09 2.23 NA NA 3.14 3.14 9.24 NA 0 0 3 1 0 0 4 5 5 0 0 5 5 5 0 9. 0.1% C U S O 4 X NA 45.00 NA NA NA NA 45.94 NA NA NA NA 28.50 NA NA NA + 2% urea a NA 0.0 NA NA NA NA 2.71 NA NA NA NA 7.68 NA NA NA n 0 2 0 0 0 0 5 0 0 0 0 5 0 0 0 10. 0.2% CuS0 4 X NA 20.00 NA NA NA NA 47.25 NA NA NA NA 28.44 NA NA NA + 2% urea a NA 0.0 NA NA NA NA 13.27 NA NA NA NA 3.59 NA NA NA n 0 1 0 0 0 0 5 0 0 0 0 4 0 0 0 APPENDIX E.2. (cont'd) Treatment 1980 1981 1982 Si t e 1 2 3 4 5 1 2 3 4 5 1 2 3 11. 4% FeS0 4 x + 2% urea a n 12. 1% CUSO4 x + 4% FeSO^ a + 2% urea n 13. 0.1% CUSO4 x + 2% FeS0 4 a + 2% urea n 14. 0.2% CUSO4 x + 2% FeS0 4 o + 2% urea n 336.25 287.50 192.50 0.0 12.31 53.60 1 3 4 NA - NA NA - NA 0 0 0 NA 138.75 NA NA 10.61 NA 0 2 0 NA 183.75 NA NA 0.0 NA 0 1 0 285.42 210.50 240.00 56.02 51.28 79.26 3 5 5 315.50 NA NA 0.0 NA NA 1 0 0 NA NA NA NA NA NA 0 0 0 NA NA NA NA NA NA 0 0 0 233.50 123.25 222.75 93.17 27.31 69.49 5 5 5 279.06 NA 287.00 104.76 NA 77.15 4 0 5 196.25 NA NA 76.72 NA NA 5 0 0 176.75 NA NA 78.88 NA NA 5 0 0 178.00 46.25 36.50 44.89 3.42 4.18 5 5 5 NA NA 29.06 NA NA 2.37 0 0 4 NA NA 32.00 NA NA 5.63 0 0 5 NA NA 33.75 NA NA 3.64 0 0 5 38.50 33.00 89.50 14.93 5.42 24.54 5 5 5 NA 26.25 NA NA 7.23 NA 0 5 0 NA NA NA NA NA NA 0 0 0 NA NA NA NA NA NA 0 0 0 NA = Treatment not applied. - = Missing value. x = Average concentration value, o = Standard deviation, n = Number of samples. ON ON APPENDIX E.3. MEAN VALUES FOR FOLIAR TOTAL IRON CONCENTRATION (PPM) OF LODGEPOLE PINE AND DOUGLAS-FIR IN RELATION TO TREATMENTS (MAIN TRIAL). Treatment 1980 1981 1982 S i t e 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 x 72.21 46.71 56.57 51.79 61.07 55.93 43.21 30.71 31.85 38.79 35.36 24.07 31.43 35.36 49.50 1. Control o 7.06 13.17 7.38 7.75 6.96 9.73 13.67 6.47 1.46 4.10 9.65 1.92 5.16 14.23 10.34 n 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 x NA - 56.75 111.07 NA NA 56.47 36.57 38.13 NA NA 25.14 35.86 31.36 NA 2. 1% CuS0 4 o NA - 5.28 36.29 NA NA 24.14 7.55 7.22 NA NA 3.69 6.47 7.36 NA n 0 0 4 3 0 0 3 5 5 0 0 5 5 5 0 x NA 47.41 NA 49.11 NA NA 44.57 NA 32.57 NA NA 26.25 NA 27.15 NA 3. 0.2% C11SO4 a NA 2.72 NA 10.36 NA NA 14.11 NA 5.40 NA NA 1.39 NA 2.01 NA 0 4 0 4 0 0 5 0 5 0 0 5 0 5 0 n x NA 48.33 NA 56.34 NA NA 39.71 NA 40.28 NA NA 28.14 NA 29.86 NA 4. 0.1% C11SO4 o NA 15.09 NA 17.41 NA NA 20.83 NA 12.02 NA NA 3.80 NA 4.54 NA n O 3 0 4 0 0 5 0 5 0 0 5 0 5 0 x 259.21 241.07 161.93 236.66 235.93 270.43 155.50 117.43 132.64 219.43 40.28 22.50 46.43 36.50 116.93 5. 4% FeS0 4 a 42.55 72.66 35.71 67.30 62.79 107.58 24.92 15.92 41.98 51.04 8.38 5.67 9.42 5.95 15.93 n 5 5 5 3 5 5 5 5 5 5 5 5 5 5 5 APPENDIX E.3. (cont'd) Treatment 1980 1981 1982 Site 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 X NA 152.77 NA 116.00 NA NA 149.57 NA 102.14 NA NA 29.71 NA 33.29 NA 2% FeS0 4 0 NA 23.20 NA 36.18 NA NA 52.56 NA 48.04 NA NA 11.23 NA 5.71 NA n 0 4 0 5 0 0 5 0 5 0 0 5 0 5 0 X 66.43 49.28 42.12 50.64 52.00 53.79 38.70 30.29 41.29 37.14 32.64 27.57 27.11 28.78 46.: 2% urea a 19.50 9.51 4.20 6.56 6.95 11.06 7.20 5.25 13.18 9.25 2.54 4.53 1.69 2.58 i i . ; n 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 1% CuS0 4 X NA _ 40.95 39.86 NA NA 94.37 30.22 38.93 NA NA 23.29 32.64 32.07 NA + 2% urea a NA - 4.32 0.0 NA NA 30.01 4.41 6.64 NA NA 5.25 4.25 8.16 NA 0 0 3 1 0 0 4 5 5 0 0 5 5 5 0 0.1% CuS0 4 X NA 66.18 NA NA NA NA 36.16 NA NA NA NA 25.00 NA NA NA + 2% urea a NA 0.86 NA NA NA NA 5.32 NA NA NA NA 5.25 NA NA NA n 0 2 0 0 0 0 5 0 0 0 0 5 0 0 0 0.2% CuS0 4 X NA 25.48 NA NA NA NA 42.50 NA NA NA NA 20.90 NA NA NA + 2% urea a NA 0.0 NA NA NA NA 22.51 NA NA NA NA 2.36 NA NA NA n 0 1 0 0 0 0 5 0 0 0 0 4 0 0 0 APPENDIX E.3. (cont'd) Treatment 1980 1981 1982 Site 1 2 3 4 5 1 2 3 4 5 . 1 2 3 11. 4% FeS0 4 X 396.43 261.54 243.12 347.27 226.79 279.71 200.28 123.57 234.36 231.85 38.64 29.57 50.50 46.00 117.1 + 2% urea 0 0.0 11.90 48.74 49.99 45.69 79.27 74.90 42.50 71.35 47.38 6.29 5.97 2.31 5.74 33. n 1 3 4 3 5 5 5 5 5 5 5 5 5 5 5 12. 1% CuS0 4 X NA - NA 325.71 NA NA 200.18 NA 302.78 NA NA 23.57 NA 44.22 NA + 4% FeS0 4 a NA - NA 0.0 NA NA 38.51 NA 85.34 NA NA 1.78 NA 11.03 NA + 2% urea n 0 0 0 1 0 0 4 0 5 0 0 4 0 5 0 13. 0.1% CuS04 - NA 156.43 NA NA NA NA 161.21 NA NA NA NA 27.36 NA NA NA + 2% FeS0 4 a NA 12.63 NA NA NA NA 64.69 NA NA NA NA 6.66 NA NA NA + 2% urea n 0 2 0 0 0 0 5 0 0 0 0 5 0 0 0 14. 0.2% CuS04 X NA 185.00 NA NA NA NA 184.43 NA NA NA NA 22.22 NA NA NA + 2% FeS0 4 a NA 0.0 NA NA NA NA 37.33 NA NA NA NA 2.15 NA NA NA + 2% urea n 0 1 0 0 0 0 5 0 0 0 0 5 0 0 0 NA = Treatment not applied. - = Missing value. x = Average concentration value, a = Standard deviation, n » Number of samples. ON APPENDIX E.4. MEAN VALUES FOR FOLIAR NITROGEN CONCENTRATION (Z) OF LODGEPOLE PINE AND DOUGLAS-FIR IN RELATION TO TREATMENTS (MAIN TRIAL). Treatment S i t e 1980 3 4 5 1981 3 4 5 1982 3 1 2 1 2 1 2 4 5 X 0.91 0.86 0.95 1.01 0.76 0.94 0.94 0.95 1.06 0.76 1.07 1.07 1.06 1.09 0.88 1. Control 0 0.03 0.06 0.08 0.07 0.12 0.05 0.07 0.11 0.10 0.10 0.04 0.04 0.12 0.06 0.07 n 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 X NA - 1.01 1.16 NA NA 1.15 1.25 1.40 NA NA 1.03 1.01 1.06 NA 2. 1Z C11SO4 0 NA - 0.09 0.03 NA NA 0.26 0.06 0.11 NA NA 0.20 0.06 0.02 NA a 0 0 4 3 0 0 3 5 5 0 0 5 5 5 0 I NA 1.01 NA 1.05 NA NA 1.18 NA 1.18 NA NA 1.06 NA 1.14 NA 3. 0.2Z &1SO4 a NA 0.07 NA 0.07 NA NA 0.04 NA 0.13 NA NA 0.16 NA 0.09 NA n 0 4 0 4 0 0 5 0 5 0 0 5 0 5 0 X NA 1.08 NA 1.01 NA NA 1.25 NA 1.16 NA NA 1.12 NA 1.20 NA 4. 0.1Z CUSO4 0 NA 0.04 NA 0.07 NA NA 0.07 NA 0.10 NA NA 0.12 NA 0.12 NA n 0 3 0 4 0 0 5 0 5 0 0 5 0 5 0 X 1.06 0.98 1.11 0.96 0.86 • 1.26 1.33 1.39 1.26 0.91 1.15 0.96 1.06 1.13 0.98 5. 4Z FeS04 a 0.08 0.13 0.03 0.03 0.08 0.09 0.14 0.13 0.10 0.05 0.13 0.11 0.14 0.07 0.06 n 5 5 5 3 5 5 5 5 5 5 5 5 5 5 5 APPENDIX E.4. (cont'd) Treatment 1980 1981 1982 Site 1 2 3 4 5 1 2 3 4 5 1 2 3 X NA 0.98 NA 1.10 NA NA 1.18. NA 1.24 ' NA NA 1.11 NA 1.09 NA 6. 2% FeS0 4 a NA 0.06 NA 0.09 NA NA 0.06 NA 0.10 NA NA 0.06 NA 0.10 NA n 0 4 0 5 0 0 5 0 5 0 0 5 0 5 0 X 0.96 0.98 1.03 1.02 0.80 1.37 1.26 1.19 1.14 0.93 1.07 1.09 0.96 1.12 0.' 7. 2% urea a 0.10 0.06 0.06 0.08 0.06 0.19 0.13 0.07 0.10 0.06 0.17 0.07 0.06 0.11 0.1 n 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 8. 1% CuS0 4 X NA - 0.94 0.96 NA NA 1.29 1.50 1.31 NA NA 0.96 1.11 0.93 NA + 2% urea a NA - 0.06 0.0 NA NA 0.26 0.20 0.06 NA NA 0.11 0.16 0.07 NA 0 0 3 1 0 0 4 5 5 0 0 5 5 5 0 9. 0.1% CuS04 X NA 1.02 NA NA NA NA 1.15 NA NA NA NA 1.01 NA NA NA + 2% urea a NA 0.06 NA NA NA NA 0.13 NA NA NA NA 0.07 NA NA NA n 0 2 0 0 0 0 5 0 0 0 0 5 • 0 0 0 10. 0.2% CuS0 4 X NA 1.14 NA NA NA NA 1.26 NA NA NA NA 1.21 NA NA NA + 2% urea 0 NA 0.0 NA NA NA NA 0.16 NA NA NA NA 0.21 NA NA NA n 0 1 0 0 0 0 5 0 0 0 0 4 0 0 0 APPENDIX E.A. (cont'd) Treatment 1980 1981 1982 Site 1 2 3 4 5 1 2 3 4 5 1 2 3 11. 4% FeSO^ X 0.94 1.03 1.04 1.15 0.86 1.50 1.38 1.40 1.39 0.90 1.14 1.06 1.06 1.09 0.! + 2% urea a 0.0 0.08 0.07 0.16 0.09 0.19 0.05 0.13 0.17 0.07 0.25 0.26 0.08 0.21 0.1 n 1 3 4 3 5 5 5 5 5 5 5 5 5 5 5 12. 1% CuS04 X NA - NA 1.08 NA NA 1.45 NA 1.51 NA NA 0.99 NA 1.09 NA + 4% FeS04 0 NA - NA 0.0 NA NA 0.29 NA 0.08 NA NA 0.09 NA 0.08 NA + 2% urea n 0 0 0 1 0 0 4 0 5 0 0 4 0 5 0 13. 0.1% CUSO4 X NA 0.99 NA NA NA NA 1.22 NA NA NA NA 1.09 NA NA NA + 2% FeS0 4 a NA 0.09 NA NA NA NA 0.11 NA NA NA NA 0.14 NA NA NA + 2% urea n 0 2 0 0 0 0 5 0 0 0 0 5 0 0 0 14. 0.2% CUSO4 X NA 0.91 NA NA NA NA 1.14 NA NA NA NA 1.12 NA NA NA + 2% FeS04 0 NA 0.0 NA NA NA NA 0.14 NA NA NA NA 0.22 NA NA NA + 2% urea n 0 1 0 0 0 0 5 0 0 0 0 5 0 0 0 NA = Treatment not applied. - = Missing value. x • Average concentration value, o = Standard deviation, n = Number of samples. 

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