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

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INVESTIGATION OF THE ZINC AND MANGANESE STATUS OF SOME STANDS TSUGA HETEROPHYLLA IN BRITISH COLUMBIA by ROBERT GADZIOLA (B.Sc. F., University of Toronto, 1981) A.THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Forestry) We accept this thesis as conforming to the standard THE UNIVERSITY OF BRITISH COLUMBIA November 1991 0 Robert Gadziola, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of \~c ^g,S^V^Y — The University of British Columbia Vancouver, Canada DE-6 (2/88) ii ABSTRACT Western hemlock has lower foliar Zn and higher foliar Mn concentrations compared to some other conifers. Existing foliar diagnostic norms for conifers imply a Zn deficiency and possibly a Mn toxicity in many stands of western hemlock. This study was undertaken in order to determine the significance of these foliar levels in the nutrition of western hemlock. The nutrition of hemlock was studied using comparative nutrition and fertilizer screening trials. The screening trials consisted of treatments of Zn and Mn<applied as foliar sprays and as soiltreatments. Different methods of application were utilized to determine if factors of the plant such as uptake and/or translocation could account for the characteristic foliar zinc and manganese levels in hemlock. In addition, a treatment was applied consisting of a complete fertilizer without Zn and Mn. This "complete-Zn-Mn" treatment was included to investigate the possibility of additional nutrient deficiencies and/or toxicities. In a comparison of total foliar concentrations, hemlock had lower Zn compared to Douglas-fir, amabilis fir and white pine. In contrast, hemlock had higher Mn compared to Douglas-fir, -amabilis fir, white pine, red cedar and yellow cedar. Analysis of cellular fractions of foliage produced two results. First, Zn accumulated in the mitochondrial fraction and Mn accumulated in the ribosomal and vacuolar fraction, Irrespective of the level of the treatment or the species. iii Accumulation in certain fractions may indicate a physiological need in that fraction or a tolerance mechanism. Second, comparing hemlock to Douglas-fir, total Zn levels tend to be consistent with levels in different fractions, indicating that total levels may be an adequate indication of physiologically active levels. But comparing hemlock to Douglas-fir, total Mn levels are not consistent with Mn levels in different fractions, indicating that total Mn levels may not be an adequate indication of physiological levels. In the fertilizer screening trials, nutrient uptake and growth responses were dependent upon the site, the level of fertilizer application, and the time since application. Nutrient uptake and positive growth responses were obtained with foliar treatments of Zn and soil treatments of Mn in both the first year and second year following fertilization. Nutrient and growth responses to soil Zn treatments were delayed until the second year following fertilization. Additional evidence supporting a Zn deficiency was indicated by a positive relationship between foliar Zn and height increment, evidence of retranslocation of Zn to new foliage in the second year following foliar Zn treatment, and the high ranking of Zn in the vector analysis from the "complete-Zn-Mn"treatment. Positive growth responses to the "cooplete-Zn-Mn" treatment were obtained in the first and second years following treatment. iv Ranking of nutrient response vectors using relative values indicated the existence of other nutrient deficiencies, besides Zn and Mn. Growth response, as measured by shoot increment ratio, was obtained primarily in the second year after treatment with foliar applications of Zn. Shoot increment ratio response occurred to soil Mn treatments in the first year of treatment. For the "complete-Zn-Mn" treatmentthere was an increase in shoot increment ratio in both the first and second years following treatment. Height increment ratio increased in response to foliar Zn applications in the second year, and to soil Mn treatments in the first year. Foliar Zn and foliar N were positively correlated with each other. Foliar Zn concentrations increased as a result of soil applications of Mn,- but applications of Zn had no effect on Mn uptake. Therefore, there was no evidence In this study to suggest that low foliar levels of Zn in hemlock are due to a Mn antagonism. The only interaction obtained with the "complete-Zn -Mn" treatment was a synergism: it caused an increase of foliar Zn. Ingestad's nutrient ratios were calculated for the foliar -levels -from the control and the "complete-Zn-Mn" treatments. V Comparing these ratios to the optimum revealed that most of the nutrients were in balance except for Fe and Mn. Existing diagnostic norms for Zn appear to adequately describe the Zn nutrition of hemlock'. Response to fertilization occurred with control foliar Zn concentrations for hemlock being below the critical level of 15 pg g~x. Diagnostic norms for Mn need to be revised. Response occurred-even though control foliar Mn concentrations for hemlock were well above the critical level of 25 ug g~v. Therefore, total foliar manganese may not be indicative of the physiological manganese status of hemlock. These results for hemlock are discussed in light of existing knowledge from the literature regarding the nutrient strategy of metal tolerant plants and low nutrient adapted plants. vi TABLE OF CONTENTS Page ABSTRACT ii TABLE OF CONTENTS vLIST OF TABLES x LIST OF FIGURES ....xiii LIST OF APPENDICES xv ACKNOWLEDGEMENTS xviCHAPTER 1. INTRODUCTION 1 CHAPTER 2 . LITERATURE REVIEW . . 4 A. Nutritional Characteristics of Western Hemlock 4 B. Zinc and Manganese Levels in Plants 9 C. Nutritional Differences Between Plants. 14 D. Deficiency and Toxicity Levels for Zinc and Manganese. . 20 E. Nutrient-Growth Models 22 1. Classical Growth Response Curve.. 22 2 . - Ingestad's Nutrient Flux Density Model....24 F. Diagnosis of Nutrient Status and Requirements ..29 1. Plant and Soil Analyses......... 29 2. Foliar Analysis 30 (a) Background(b) Physiological and Empirical Basis of Foliar Analysis 31 (c) Nutrient Diagnosis Using Foliar Analysis 33 (i) Total Analysis with Single Nutrients . 33 vii Page (ii) Total Analysis using Nutrient Balances 40 (iii) Physiological Active Fractions.... 44 G. CONCLUSIONS .... 48 CHAPTER 3. METHODS AND MATERIALS.. 51 A. Site Description1. Location of Study Areas 51 2. Stand Characteristics 2 3. Soil Characteristics....... .52 B. Experimental Design 54 C. Fertilizer Treatments... ....59 D. Field Sampling 60 E. Chemical Analysis 3 F. Scanning Electron Microprobe 65 G. Soil Sample Preparation and Analysis. 66 H. Measurement of Fertilizer Response... ...67 I. Statistical Analysis... 69 CHAPTER 4. RESULTS ..71 A. Comparative Nutrition.......... ...71 1. Total Levels .72. Extractable Zinc and Manganese 71 3. Cellular Fractions of Foliage 73 B. Fertilization Experiments .. 79 1. Nutrient and Growth Responses 79 (a) Zinc 83 (i) Foliar Zinc Treatments 83 viii Page (ii) Soil Zinc Treatments 87 (iii) Comparison of Foliar Versus Soil Treatments 90 (b ) Manganese 9(i) Foliar Manganese Treatments ...90 (ii) Soil Manganese Treatments 93 (c) Complete-Zn-Mn 97 (i) Nutrient and Growth Responses ... 97 2. Shoot Increment Ratio. 103 3. Height Increment Ratio 108 C. Retranslocation.......... ....108 D. Nutrient-Growth. Interactions , 114 E. Nutrient Interactions 117 1. Interactions with Zinc 117 (a) Manganese 117 -; (b) Nitrogen 117 2. Interactions with Manganese 122 (a) Zinc 12F. Relationship of Response to Site 126 CHAPTER 5. DISCUSSION 131 A. Comparative Nutrition 131. Total Levels 131 2. Extractable Zn and Mn 133 3. Cellular Fractions 134 B. Fertilization Experiments 135 1. Nutrient and Growth Responses 135 (a) Zinc 136 ix Page (i) Comparison of Foliar Versus Soil Treatments ..136 (ii) Zinc Tolerance 140 (b) Manganese..... -....143 (i) Comparison of Foliar Versus Soil Treatments .144 (ii) Toxicity Levels 146 (ill) Manganese Requirements .......148 (iv) Manganese Tolerance. . . .. 149 C. Comparison and Consideration of Adequate Levels 150 1. Individual Nutrients.. 152. Nutrient Balance 156 D. Retranslocation. . . . .159 E. Nutrient Interactions 160 F. Foliar Application of Zinc in Forestry.......165 CHAPTER 6. CONCLUSIONS ..166 LITERATURE CITED. »v . . . .  . . .173 APPENDICES vl88 X LIST OF TABLES 1. Comparison of surface soil and forest floor properties under Douglas-fir and western hemlock stands from coastal versus Cascade sites. 7 2. Tissue micronutrient levels in Pacific Northwest conifers 10 3. Foliar Zn/Mn levels for various tree species occurring in the same stands located in the interior of B.C . .13 4. Linear correlations between growth response and current needle N concentration, N content and dry weight in the two years following fertilization. ... ; .34 5. General representation of changes in total content, yield and concentration as affected by imposed treatments ... ....38 6. Chemical characteristics of the soil profiles.. 55 7. Treatment-levels used in the fertilizer trials with the number used in the text and its corresponding control.....56 8. Total foliar Zn, Mn and Fe, water-soluble Zn and Mn, and active Fe for different species in the same stands........ 72 9. Equations and Ra values for the height increment versus foliar shoot-' per mass relationships. ...... . . . . 82 10. Foliar zinc nutrient response and foliar mass per shoot response to foliar applications of zinc in the first and second years following treatments ..84 11; Foliar zinc and foliar mass per shoot response to soil applications of zinc in the second year . . 88 12. Foliar manganese and foliar mass per shoot response to soil treatments of manganese. ........94 13. Nutrient response to complete-Zn-Mn treatment... 98 14. Shoot increment ratio growth response to zinc ...104 15. Shoot increment ratio response to soil applications of manganese in the first year (1986) on sites 4 and 5. 106 xi Table Page 16. Shoot increment ratio response to the complete-Zn-Mn treatment 107 17. Height increment ratio response.......... 109 18. Change in foliar zinc concentration of current year's foliage with time and age of foliage following treatment for the low zinc foliar treatment (4) on sites 1 and 2 Ill 19. Change in foliar zinc concentrations of current year's foliage with time and age of foliage following treatment for the high zinc foliar treatment (5) on sites 1 and 2 .112 20. Change in foliar manganese concentration of current year's foliage with time and age of foliage following treatment for the manganese soil treatment (3) on sites 1 and 2 113 21. Equations and Ra values for the height increment vesus foliar zinc relationships. 116 22. Foliar manganese response to soil and foliar applications of zinc in the first year (1986) on site 5 118 23. Equations and H' values for the foliar nitrogen versus foliar zinc relationships...... 120 24. Foliar zinc response to soil and foliar manganese applications ...123 25. Foliar zinc response in the first year (1986) to soil applications of manganese on site 5 124 26. Summary of nutrient and- growth responses to foliar zinc treatments 127 27. Summary of nutrient and growth responses to soil zinc treatments. 8 28. Summary of nutrient and growth responses to soil manganese treatments ....129 '29. Summary of nutrient and growth responses to the complete -Zn-Mn treatment.. 130 30. Soil data for all sites. 132 31. Effect of solution pH on micronutrient concentrations in Douglas-fir ari*d western hemlock roots and needles 142 xii Table Page 32. Ingestad's foliar nutrient ratios for the complete-Zn -Mn and control treatments on sites 4 and 5 for the years 1986 and 1987.. . . . 157 xiii LIST OF FIGURES Figure Page 1. Factors of the plant which affect plant nutrition 15 c 2. The three models of plant-soil relationships............ 19 3. Relationship between plant growth and tissue nutrient concentrations. 23 4. The relationship between the external nutrient supply and relative growth rate ... .. ... 26 5. Schematic -representation of a detailed mechanistic model of tree growth.......... 32 6. Vector method for the interpretation of nutrient -growth response data using nutrient concentration, nutrient content and dry mass of needles 37 7. Example of a boundary line.... ...39 8. Diagramatic representation of crop response to a number of limiting factors...... 41 9. Location of the study plots in the Lower Hainland.......52 10* Zinc concentrations in different cellular fractions from the current year's foliage (1986) of the control treatment and high foliar Zn treatment from site 5 .75 11. > Manganese concentrations in different cellular fractions from the current year's foliage (1986) of the control treatment and high soil Mn treatment from site 5 76 12. Manganese concentrations in different cellular fractions from the current year's foliage in 1987 of different species on site 5 .....77 13. Zinc concentrations in different cellular fractions from the current year's foliage in 1987 of different species on site 5 78 14. Scatter plots of height increment versus the foliar mass per-shoot for site 1 inthe first and second years following treatment .81 15. Vector diagrams of growth response to foliar applied zinc in the first year on site 2, in the second year on site 2, in the second year on site 3, and in the first year on site 5 86 xiv Figure Page 16. Vector diagram of the second year growthresponse to soil applied zinc on site 4 for foliar zinc ..89 17. Nutrient efficiency of foliar Zn versus soil Zn treatments in supplying the plant with Zn .91 18. Vector diagrams of the second year growth response to foliar applied manganese on sites 4 and 5 for foliar manganese. ....92 19. Vector diagram of the growth response to soil applied Mn for foliar Mn in the second year on site 4 and in the first year on site 5... 96 20. Vector diagram of the first year growth response to the complete-Zn-Mn treatment on site 4. 101 21. Vector diagrams of the first and second year growth response to the complete-Zn-Mn treatment on siteS 102 22. Scatter plots of height increment versus current year's foliar zinc levels in the first year and in the second year site 4 115 23. Scatter plots of foliar nitrogen versus foliar zinc in the first and second years from site 1 119 24. Scatter plot of total soil nitrogen versus extractable soil zinc both from the forest floor for all sites..... 121 25. Vector diagram of first year nutrient response of zinc to soil treatments of manganese from site 5... 125 26. Possible pathways of solute movement through the leaf..145 27. Ribbon model of a single zinc finger domain (ADRla) incorporating tetrahedral coordination of zinc by cystine (C) and histidine (H) 164 XV LIST OF APPENDICES Appendix Page A.l Site and Soil Description of Site 1 ....188 A.2 Site and Soil Description of Site 2 .....190 A.3 Site and Soil Description of Site 3 192 A.4 Site and Soil Description of Site 4... . . . .194 A. 5 Site and Soil Description of Site 5 196 B. 1 Modified Parkinson and Allen Digestion for Plant Tissue Analysis . 198 B.2 Nitric Acid Digestion for Analysis of Copper and Iron in Foliage . . 199 B.3 Procedure for the Determination of Sulphate-Sulphur in Foliage 200 B.4 Determination of Active Iron in Foliage. 201 B.5 Determination of Extract able Zinc . . 202 B. 6 Determination-of Extractable Manganese from Foliage.... 203 C. Comparison of Foliar Zn, Mn, and Fe Concentrations Using AA and ICP....... ... 204 D. Formulae Used to Convert Foliar Zn and Mn Concentrations Measured on the AA to Corresponding ICP Values > 205 E. Comparison of the- Recovery of Foliar Nutrients from AA and ICP 206 F. Preparation and Analysis of Cellular Fractions from Foliage 8 G. Fixation and Embedding Procedure 209 H. The Mehlich 3 Soil Extraction Method 210 I. Foliar nutrient guidelines for the interpretation of nutritional status 212 J. Foliar Nutrient Data for the Cellular Fractions........217 K.l Scatter plot of the 1986 height increment versus the 1986 foliar mass per shoot for site 2. .... .219 xvi Appendix Page K.2 Scatter plot of the 1986 height Increment versus the 1986 foliar mass per shoot for site 3. 220 K.3 Scatter plot of the 1986 height increment versus the 1986 foliar mass per shoot for site 4... ....221 K.4 Scatter plot of the 1987heightIncrement versus the 1987 foliar mass per shoot for site 4.. 222 K.5 Scatter plot of the1986 height increment versus the 1986 foliar mass per shoot for site 5 223 R.6 Scatter plot of the 1987 height increment versus the 1987 foliar mass per shoot for site 5 224 "L. Foliar Nutrient Data from the Fertilization Trials..... 225 M.i Foliar Nutrient Data from Site 1 for the Study of Zn and Mn Retranslocation with time and age...............264 M.2 Foliar Nutrient Data from Site 2 for the Study of Zn and Mn Retranslocation with time and age 265 M.I Scatter plot of first year total height increment in 1986 versus first year foliar zinc in 1986 for site 5. .266 N.2 Scatter plot of second year total height increment in 1987 versus foliar zinc levels in 1987 for site 5 267 .0.1 Scatter plot of foliar nitrogen versus foliar zinc for current year's foliage in 1985 for site 2. ...268 0.2 Scatter plot of foliar nitrogen versus foliar zinc for -current year's foliage in 1986 for site 2. 269 0.3 Scatter plot of foliar nitrogen versus foliar zinc for current year's foliage in 1986 for site 3 ..270 0.4 Scatter plot of foliar nitrogen versus foliar zinc for current year's foliage in 1986 for site 4. 271 0.5 Scatter plot -of foliar nitrogen versus foliar zinc for current year's foliage in 1987 for site 4. .272 0.6 Scatter plot of foliar nitrogen versus foliar zinc for current year's- foliage in 1986 for site 5....... 273 0.7 Scatter plot of foliar nitrogen versus foliar zinc for current year's foliage in 1987 for site 5. ..274 xvii ACKNOWLEDGEMENTS I am appreciative to my supervisory Dr. T. M; Ballard for providing the initial idea for this thesis, and for his support and effort in bringing this work to its fruition. I am also grateful to the other members of my Supervisory Committee: Dr. A. A. Bomke, Dr. Ki Klinka, and Dr. 6. F. Weetman for the time and effort they took to review and provide constructive criticism of this thesis. The contribution of Dr. R. J. Zasoski is acknowledged for the contribution he provided as the External Examiner; In producing this thesis I wish to acknowledge the contributions made by Mr. R. Carter, Mrs. R. Lowe, Ms. E. Wolterson, and Mr. B. Von Spindler. I would also like to thank the staff of -the Mission Tree Farm (Mission, B.C.), and the U.B.C. Research Forest (Maple Ridge, B.C.) for providing me with the facilities to establish fertilizer trials. I would like to thank the various organizations which provided me with the financial support which allowed me to attend graduate school and conduct this research. These were the Canadian Forestry Service, the Natural Sciences and Engineering Research' Council of Canada, Fletcher Challenge of Canada Ltd., the Faculty of Forestry, and the Department of Soil Science. xviii In addition, the friendships provided by fellow students and staff in the- Department of Soil Science and Faculty of Forestry deeply enriched my life. Finally, I am thankful to my family for their- love, support and encouragement. I dedicate this thesis in memory of my Father., Leon Peter Gadziola. 1 CHAPTER 1. INTRODUCTION The species Tsuga heterophylla (Raf.) Sarg. (western hemlock) characteristically has lower foliar zinc (Zn) and higher foliar manganese (Mn) concentrations relative to some other conifer species throughout British Columbia and the United States Pacific Northwest. Curiosity about this phenomena motivated this research. The patterns of foliar Zn and Mn levels in hemlock are of practical significance in forestry. Attempts to increase the productivity of hemlock with nitrogen fertilizers have met with variable and inconsistent results. This has lead to interest in the status of other nutrients in hemlock such as Zn and Mn. . In comparison to suggested critical foliar levels for some B.C. conifers, hemlock often has foliar-Zn. levels which fall into the low to possibly deficient zone (being less than the critical foliar Zn level of 15 ug g~x), and has foliar Mn levels which fall into the high to possibly toxic range (being higher then the critical foliar Mn level of 25 ug g~*). Therefore, the foliar Zn and Mn concentrations characteristic of hemlock suggest that Zn is at possibly deficient and Mn is at possibly toxic levels. Up to the present time the patterns of foliar Zn and Mn concentrations of hemlock have been described but no work has been done on their significance in the nutrition of hemlock. 2 Therefore, the objective of this study was to investigate the significance of the pattern of foliar Zn and Mn concentrations in the nutrition of hemlock. The specific questions asked were: Is Zn sometimes deficient and is Mn sometimes toxic for the growth of hemlock? Are the lower foliar Zn concentrations due to a Mn antagonism? Are the lower foliar Zn and higher foliar Mn concentrations of hemlock compared to some other conifers due to factors of the plant such as uptake and/or translocation or factors of the soil such as fertility? Are there additional nutrient deficiencies in hemlock? A comprehensive literature review was made to learn what was already known about hemlock nutrition, Zn and Mn levels in plants* plant factors affecting nutrition, deficiency and toxicity levels for Zn and Mn, nutrient-growth relationships, and methods of nutrient diagnosis. Two approaches were taken in this research. The first step involved a comparison of Zn and Mn nutrition among several conifer species, to check the premise for this study. This led to another question: although plants may have different total foliar Zn and Mn concentrations do they have similar physiological levels? A review of the literature suggests some possible mechanisms, and some experimental work (extractable and active nutrient levels, and foliar Zn and Mn distributions between cellular fractions) was directed to this question. The second step involved the use of fertilizer screening trials, 3 using foliar and soil treatments of Zn and Mn, and a soil application of a "complete-Zn-Mn" treatment. By measuring nutrient and growth responses, inferences could be made about deficiencies and toxicities. Nutrient retranslocation, nutrient interactions, and nutrient-growth relationships could also be examined using data from the fertilizer trials. Since the patterns of Zn and Mn foliar concentrations are found over a wide area of hemlock's range, the sites selected for the fertilizer trials did not cover a wide range of ecosystem conditions. In addition, the enormity of collecting several hundred samples, each of which would be subjected to several chemical analyses, and the requirement of having sites easily accessible from the University of British Columbia to facilitate the work, limited' the number of sites which could be investigated. These constraints obviated using data and conclusions to generalize about hemlock in the region as a whole. However, the site selection proved adequate for its purpose: it succeeded in finding hemlock stands low in Zn and high in Mn suitable for testing hypotheses about deficiency, toxicity and antagonism. 4 CHAPTER 2. LITERATURE REVIEW A. Nutritional Characteristics of Western Hemlock Western hemlock occurs in five biogeoclimatic zones of B. C. It grows as a climax species in the Coastal and Interior Western Hemlock zones. In the Coastal Douglas-Fir zone, it develops- as a climax species only in subhydric habitats. In the subalpine Mountain Hemlock and Engelmann Spruce-Subalpine Fir zones, the short vegetative season is the major obstacle which limits 'the distribution and growth of western hemlock (Krajina et al. 1982). Western hemlock is commonly associated with strongly podzolized soils and these soils characteristically have a mor humus layer which has a low rate of mineralization. This suggests that hemlock is tolerant of acidic soils and requires lower quantities of nutrients compared to some other trees (Krajina er al. 1982). The productivity of hemlock was found by Lowe and Klinka (1981) to have a lower negative correlation with the percent yield of soil lipids of the humus layer as compared to Douglas-fir. Lipids accumulate to high levels in - soils where biological activity is inhibited (Lowe and Klinka 1981), which indicates a negative relationship between the abundance of soil lipids and mineralization. The lower correlation between the amount of soil lipids and the productivity of hemlock suggests that hemlock is less sensitive to low rates of decomposition, 5 mineralization and nutrient release compared to Douglas-fir (Lowe and Klinka 1981). The productivity of hemlock was also positively correlated to the pyrophosphate-extractable Fe+Al (PFeAl) and carbon content in the B horizon (TCB). Both the PFeAl and TCB are Indices of the extent and intensity of podzol formation. This helps to explain the occurrence of hemlock on podzolic soils. Hemlock regeneration flourishes on rotten logs, stumps or mineral soil exposed on trails or on mounds and pits created by windthrown trees (Christy et al. 1982; Stewart 1989). The organic layer is frequently a substratum for hemlock even above the more base-rich, high pH soil layers in which this tree does •not regenerate (Krajina et al. 1982). Rotten wood is an important substrate-for mycorrhizal associations of moist hemlock habitat types in the northern Rockies (Harvey er al. 1979). More than 95% of the active primary roots of hemlock have been found to be mycorrhizal (Gill- and Lavender 1983). Since mycorrhizae -have been found to enhance; nutrient uptake in many species, their abundance on hemlock roots suggests that mycorrhizae play an important role in hemlock nutrition. During the first season, seedlings are able to survive on rotten wood without mycorrhizae; however, mycotrophy is advantageous for growth (Harvey er al. 1979). Two-year-old seedlings growing on mineral soil had greater growth than those growing on the rotten wood substrate. The effect of substrate on growth disappeared in the third year (Christy et al. 1982). Roots of seedlings which originally established on rotten wood eventually extended their roots into mineral soil and the original substrate disappeared. The nutritional source then shifts from woody substrate to mineral soil (Christy er al. 1982). Hemlock occurs- on soils which characteristically have low pH and a pH-dependent cation exchange capacity (CEC); often low base saturation and abundant Al on the soil exchange complex; high organic matter contents; and the presence of sesquioxides and/or "andic" soil properties (Ryan 1983). This is evident when one compares soil properties for hemlock forests versus Douglas-fir- forests (Table 1). Consequently, soluble Al and Mn are high (Toy 1984), and nitrogen is predominantly in the ammonium (NH**) form- (Haynes 1986). Anderson er al. (-1982) found that nitrification in two coastal Washington soils under hemlock stands was negligible. This was also found for forest floor and mineral soils associated with hemlock trees in northwestern Washington (Turner and Franz 1985).. Therefore, the nitrogen nutrition of hemlock appears to be adapted to the ammonium form. Nutrientstudies by Krajina er al. (1973), and van den Driessche (1971, 1976) tend to support this observation. Ryan er al. (1986a, 1986b) investigated the tolerance of seedlings of Douglas-fir, western hemlock, and western redcedar to solution acidity and Al concentration in solution cultures. Western hemlock survived and thrived in acid solution of pH 3 but the other species had greater mortality and reduced growth (Ryan 7 Table I. Comparison of surface soil and forest floor properties under second growth Douglas-fir (A) and western hemlock (B) stands from coastal (W) versus Cascade (E) sites (from Zasoski et al. (1986)). Region A B Surface Soil 0-15 cm PH E 5.1 4.5 W 4.9 4.4 Exchangeable Cations (meq per 100-8 •)• Ca E 3.8 1.7 W 3.0 1.2 Kg E 0.8 0.36 W 0.96 0.73 K E 0.41 0.27 W 0.38 0.32 CEC E 26.0 35.1 W 37.1 47.3 Base Saturation (X) E 20.4 7.2 W 11.4 4.8 Available P (pg g E 97.0 44.0 W 51.0 15.0 Total P (|ig E 1070 809 W 13 70 954 Available S (ug g -*) E 7.7 9.9 W 9.5 10.2 Total N (%) E 0.16 0.31 W 0.31 0.45 8 Table 1. (concluded). Total C (%) E 4.7 8.4 W 8.3 10.9 C:N Ratio E 30.3 28.5 W 27.2 24.4 Forest Floor Total Mass (kg ha"*) Total N (%) Total C (X) E 21700 29800 W 18700 27000 E 0.95 1.05 W 1.05 1.02 E 39.1 41.5 W 39.7 43.4 C:N Ratio E 38.7 W 39.0 38.0 42.9 9 et al. 1986a). Both western hemlock and western red cedar were found to be especially tolerant of acid-Al conditions with a solution pH of 3.5 and at the highest Al treatment of 100 ug g~l (Ryan er al. 1986b). Aluminium adversely affected the tissue concentrations of Ca and Mg. The ability of western hemlock to grow in acid-Al conditions is suggested to be related to thi6 species' physiological tolerance of excess H-cations in solution and low tissue requirements of Ca and Mg (Ryan et al. 1986a, 1986b). The specific physiological or biochemical Al tolerance mechanisms for hemlock have not been investigated. In a mature mixed subalpine stand, T. aertensiana and A. amabilis had the highest concentration of Al in the fine root component relative to all tissues analyzed (Vogt et al. 1987). It was hypothesized that accumulation in the roots is an effective mechanism for avoiding Al toxicity. The large root biomasses of these subalpine stands allow for large amounts of Al to be taken up and immobilized in roots. The high root turnover in these stands may be a result of root senescence occurring in response to high Al accumulation. Root senescence would be an effective mechanism for removing Al from the biological component (Vogt er al. 1987b). B. Zinc and Manganese Levels in Plants The concentrations of Zn and Mn in the foliage of various tree species in the Pacific Northwest are listed in Tables 2 and 3. Hemlock tends to have lower foliar Zn and higher foliar Mn 10 Table 2. Tissue micronutrlent levels (|ig g~M in Pacific Northwest conifers. Species Age Type Location Zn Mn Source Douglas-fir 13-49 field Van. Is. B.C. 17-35 452 -758 1 1 outdoors Seattle, WA 11-28 125 -785 2 4 plant Hoquiam, WA 21-32 390 -580 3 7 plant Pack For. WA 21 4 20 field Puget Sound WA 14-31 350 -2010 5 30 field Coastal B.C. 11-18 174 -465 6 30 field Coastal B.C. 23 121 6 5-32 plant Coastal B.C. 13 292 7 5-32 plant Coastal B.C. 8 316 ,. . 7 60-73 field Coastal B.C. 18 186 7 plugs nursery Seattle, WA 35 8 Sitka spruce 1 outdoors plugs nursery Western red cedar plugs nursery 27 field 25 field Ponderosa pine plugs nursery 7 plant Seattle, WA Seattle, WA Seattle, WA Coast OR, WA, B.C. Interior OR, WA Seattle, WA Pack For., 40 WA 31-68 98-403 59 24 13-26 69-383 21^-48 102-368 64 2 8 8 9 9 8 4 11 Table 2 (continued) Western hemlock 1 outdoors Seattle, 33-36 140-619 2 WA plugs nursery Seattle, 45 8 WA 23-39 field Cascades, 13-24 885-1213 10 OR 23-39 cont. Coastal 7-15 617-764 10 OR 23-39 field Cascades 15-23 736-1087 10 OR 23-39 fert. Coastal 9-15 434-586 10 OR 60-150 field Van. Is. 3-14 1580-198 1 B.C. 4 plant Hoquiam, 10-19 480-930 3 WA 1-2 lath Olympla, 0.1-3 249-696 11 WA 20-30 field Cascades 16-20 1200-190 12 WA 20-30 field Coastal 17-20 1000-110 12 WA 6-10 field Western 17-19 616-1922 13 WA 13-15 plant Ozett, WA - 7-16 14 70 field Gold River, 5 1804 15 B.C. 70 field Kaprino I, 3 1017 15 B.C. 72 field Kaprino II, I 1263 15 B.C. 52 field Island Hwy, 5 1493 15 B.C. 70 field Beaver Lake , 6 1720 15 B.C. 71 field Rupert Main » 5 1626 15 B.C. 30 field Brittain 7 1187 15 River, B.C. 5.9 1114 15 6.6 753 15 6.4 880 15 7.4 1043 15 7.7 930 15 8.8 1127 15 7.8 811 15 12 Table 2 <concluded) Western white pine -20 field Sub-alpine fir Puget 26-62 Sound, WA Lodgepole pine White spruce Douglas fir Western red cedar Spruce hybrid field field field field field field Interior, B.C. Interior, B.C. Interior, B.C. Interior, B.C. Interior, B.C. Interior, B.C. 16-64 33-71 37-76 13-27 8-12 141-1800 369-1175 245-747 154-1025 274-2244 92-384 33-66 225-637 16 16 16 16 16 16 Sources 1. Beaton er al. 1965 2. Rollwagen 1981 3. Porada 1987 4. Greenleaf-Jenkins 1985 5. Zasoski et al. 1977 6. Carter et al. 1984 7. Carter er al. 1986 8. Zasoski et al. 1984 9. Radwan and Harrington 1987 10. Gill and Lavender 1983 11. Radwan and DeBell 1980a 12. Radwan and DeBell 1980b 13. Zasoski er al. 1990 14. Zasoski et al. 1990 15. Carter unpublished 16. 'Ballard unpublished 13 Table 3. Foliar Zn/Mn levels (|ig g~M for various tree species occurring in the sane stands located in the interior of B.C. (from Ballard (personal communication)1). Species Plots Western hemlock 3/2225 Western red cedar Douglas fir 14/697 Lodgepole pine 50/662 White spruce Spruce hybrid 1/825 2/850 5/1850 2/732 11/358 11/100 11/280 14/338 53/227 61/474 Species Plots Western hemlock 3/1625 3/2425 3/1775 1/1125 5/975 Western red cedar 12/316 11/384 Spruce hybrid 47/427 Species Plots Western hemlock 1/1175 5/1275 Western red cedar 8/180 Douglas-fir 18/379 1. T. M. Ballard. Professor, Faculty of Forestry/Department of Sail Science, University of British Columbia. Unpublished Data 1984. 14 concentrations compared to other species, not only in different stands (Table 2), but also in the same stands (Table 3). In a mixed subalpine stand of A. amabilis and T. mertensiana, the two species were found to have clear differences in their ability to accumulate specific elements from the soil (Vogt et al. 1987a). Differential' nutrient-levels have also been found between other wild plant species on the same sites (Gerloff er al. 1966), and between agricultural^ plants of different species (Gladstones and Loneragan 1970; Collander 1941) and varieties when supplied with the same amounts of nutrients (Brown et al. 1972). This indicates that different species of plants have different tolerances and requirements for' nutrients, and that factors of the plants themselves affect nutrient uptake. C. Nutritional .Differences' between Plants There are a number of reasons for differences in nutrition between species and genotypes which are summarized in Figure 1. These are related to uptake, transport and utilization in the plant (Marschner 1986). Both uptake and growth are assumed to be controlled by cytoplasmic pools through feedback and substrate supply, respectively. Consequently, species differences in uptake and growth might be related to cellular compartmentation of nutrients (Chapin 1988). Species adapted to different soil fertilities generally differ in the distribution of nutrients among various chemical fractions (Chapin 1988). Species may 15 (I) Nutrient Efficiency (1) (2) a) Demand on cellular level a) Root-shoot transport (long distance) b) Utilization within the shoot (eg. retran8locatlon) W Transport within the root (short distance) c) Compartmentatlon/bindlng-form within the roots (II) Acquisition of (1) Root morphology a) Roots themselves . i) inherent il) response to deficiency b) Mycorrhizae (eg. secretions < (2) Root physiology and biochemistry a) Affinity of the uptake system (Km) b) Threshold concentration (Cmln) c) Modifications of the rhlzosphere I) Passive (eg. cation-anlon uptake) ii) Active response to deficiency chelating, reducing compounds, protons) Figure 1. Factors of the plant which affect plant nutrition (from Marschner (1986)). 16 differ in the compartmentation of nutrients into specific plant parts, chemical fractions, or cellular compartments (Chapin-1988). Foliar concentrations may reflect these differences (Bowen 1981), or some of the same fractions may have similar -nutrient concentrations for the same physiological processes. Hemlock tends to have lower nutrient requirements compared to other conifers (Krajina et al. 1982). Plants adapted to low nutrient conditions have a growth rate which is relatively insensitive to variation in the rate of nutrient supply (Chapin 1988). During periods of high nutrient availability there would be accumulation of vacuolar stores (luxury consumption). Luxury consumption of nutrients buffers the plant from variation in external nutrient supply (Chapin 1988). There is a tendency towards stable ionic composition of- the cytoplasm (Glass and Siddiqi 1984; Leigh and Jones 1986). Plants can affect nutrient uptake by affecting the pH of the rhizosphere. There are differences in the rhizosphere pH among plant species growing in the same soil (Harsc'hner 1986). Hemlock has a higher ratio of H* release / MH** uptake than Douglas-fir. This suggests hemlock may not only tolerate acid conditions but would tend to create acidity in the rhizosphere (Bygiewicz et al. 1984). Hemlock had lower mean ammonium uptake rates for both mycorrhlzal and nonmycorrhizal roots compared to Douglas-fir. However, mycorrhlzal roots enhanced ammonium uptake rates in hemlock (Rygiewicz et al. 1984). 17 The Corn of nitrogen used by a plant can have an influence on the plant's overall nutrition. Plants with annoniui nutrition tend to absorb cations in excess of anions (N being the element often absorbed in the largest amounts), with a net efflux of H* into the rhizosphere. As a consequence of ammonium nutrition there is a decrease in the uptake of cations compared to that observed with nitrate nutrition. This may be attributed to ionic competition with ammonium or with the excreted H*" ions (Haynes 1986) . The reduction in cation uptake with ammonium nutrition may be a mechanism of plant tolerance to Al and Mn on acid soils. In general, ammonium inhibits the plant's uptake of Mn and Al (Haynes 1986). Ammonium nutrition has two other consequences. Firstly, binding of free- ammonium must take place with organic compounds in order to tolerate the high ammonium levels (Kirkby and Hughes 1970). Secondly, ammonium plants must ensure a synthesis of organic anions independently of nitrate reduction as a means of compensating for the inadequate supply of anions. The smaller quantity of anions would hinder the transfer of cations to the shoots. It is through an active synthesis of organic acids that plants could have an equivalent or better growth when they depend on ammonium as a nitrogen source (Salsac er al. 1987) . 18 Baker (1981), Identified three nutrient models of plant-soil relations. These are the accumulator, the excluder and the indicator (Figure 2). Accumulators are plants where metals are concentrated in above-ground plant parts from low or high soil levels (Baker 1981). Excluders are plants where metal concentrations in the shoots are maintained constant and low over a wide range of soil concentrations, by differential uptake and transport, up to a critical soil value above which the mechanism breaks down and unrestricted transport results. Indicators are plants where uptake and transport of metals to the shoot are regulated so that internal concentration reflects external levels (Baker 1981). In both the accumulators and the excluders, the mechanisms of tolerance are largely 'internal' in that there is active detoxification of metal ions. It is the sites of detoxification which differ, being largely within the root in excluders and in the shoots in accumulators (Baker 1981). Detoxification may result from cell-wall binding, active pumping of ions into vacuoles, complexing by organic acids and possibly by specific metal-binding protein, enzymatic adaptations and effects on membrane permeability (Baker 1987). Ectomycorrhizae may play a role in metal exclusion by restricting uptake or through accumulation. Taylor (1987) made a distinction between internal tolerance and exclusion based upon the site of metal 19 Accumulator Indicator Plant Plant Soil Figure 2. The three models of plant-soil relationships. The axes represent nutrient concentrations (from Baker (1981)). 20 detoxification orimmobilization, either in the symplasm (internal) or apoplasm (exclusion) of roots. Exclusion of a metal may be by immobilization in the cell wall, exudation of chelates or organic acids from roots, a redox barrier at the plasma membrane, or a pH barrier at the plasma membrane (Taylor 1987). D. Deficiency- and Toxicity Levels for Zn and Mn The foliar nutrient concentrations tend to be an integration of all the soil and plant factors which affect nutrient availability to the plant. Therefore, they serve as an indicator of plant nutrient availability. The critical Mn deficiency level found in general for most plant foliage ranges from 15 to 25 ug g~* with the sufficient level being 20-300 ug g-*- (Kabata-Pendias and Pendias 1984). Toxicity to Mn generally occurs at a concentration above about 500 ug g-1 (Kabata-Pendias and Pendias 1984); toxicity to Mn at foliar concentrations of over 1,000 ug g~* Is common(National Academy of Sciences 1973). There is abundant evidence which suggests that Mn levels found in plants are not reflective of physiological requirements. Species as diverse as sugarbeets and wheat have a critical deficiency level in the range of 10-20 ug g~x (Clarkson 1988). In Vacciniua vitis-idaea, plants having foliar Mn levels of 6,800 21 to 12,300 ug g~x had similar rates of photosynthesis, dry natter production and number of leaves as plants containing 18 to 1,500 ug g-*- of Mn (Miller 1987). In tree species fron the temperate forests of Central Japan, Mn concentrations of the shoots vary between species by a factor of 180 (Marschner 1988). In addition, large differences have been found between barley genotypes in Mn efficiency. Therefore, differences in Mn concentrations between species growing on the same soil probably have little to do with differences in Mn requirements (Clarkson 1988) or utilization in the plants (Marschner 1988). They are probably due to differences in Mn acquisition from the soil (Marschner 1988). These differences may partially reflect the extent to which species are able to acidify the rhizosphere (Clarkson 1988) or produce Mn reducing organic root exudates. Memon and Yatazawa (1982) extracted water-soluble Mn from Mn accumulator plants. More than 70% of the total Mn was water-soluble. Results of electron probe x-ray microanalysis revealed that the portion of Mn which was water-insoluble was contained in the cell walls. The critical Zn deficiency level for plants is from 10-20 ug g"1, with the level of sufficiency being from 27-150 ug g~l, and the threshold of toxicity at 100-400 ug g~x (Kabata-Pendias and Pendias 1984). There are some reports that only a portion of the total level of zinc is physiologically active. Water-soluble Zn has been examined as an indication of the Zn status of the 22 plant. Cakmak and Marschner (1987) found water-soluble zinc to be a suitable indicator of zinc nutritional status in general. This was because of the close correlation between water-soluble zinc and visual Zn deficiency symptoms, levels of chlorophyll, superoxide disrautase, and membrane permeability. Rahimi and Schropp (1984) found water-soluble zinc to be an indicator of the Zn nutrient status of the plant because of its relationship to the activity of carbonic anhydrase. When one tries to make an accounting of all the zinc, there is a discrepancy between total and identifiable zinc (Hewitt 1983). Up to 60% of the plant zinc has been accounted for in its identifiable forms in proteins (Hewitt 1983). E. Nutrient-Growth Models 1. Classical Plant Growth Curve The classical plant nutrient-growth model is an empirical relationship described by a curve of diminishing returns. The curve may represent a relationship between plant growth and tissue nutrient concentrations (Figure 3), between plant growth and either soil nutrient concentrations or fertilizer additions. The curve consists of four zones: deficient (A-B), sufficient (B-C), luxury (C-D) and toxicity (D-E). The deficient zone may be distinguished from the adequate zone by the critical nutrient 23 Figure 3. Relationship between plant growth and tissue nutrient concentrations. 24 concentration. This is the nutrient concentration that corresponds to 90% of maximum yield. Other modifications of the curve by Dow and Roberts (1982) have a critical nutrient range rather than a critical nutrient concentration (B-C). This relationship has been expressed mathematically using the Mitscherlich model, quadratic and exponential models, and inverse polynomials and hyperbolic models (Walworth and Sumner 1988). This traditional model of plant growth-nutrient relationships has certain theoretical problems. Firstly, this type of relationship is empirical and does not lead to results of general application (Landsberg 1986). From the growth curve one attempts to define critical foliar nutrient concentrations by •establishing relationships between final yield, or growth increment over a period, and nutrient concentration in the foliage, measured at the end of that period. These lead to a second problem in that the results are highly variable because the tissue nutrient status (x) at any time (t) affects the growth rate at that time (dy/dt) (Equation 1) (Landsberg 1986). dy/dt - f(x) (I) where: y = growth t = time x = nutrient concentration 2. Ingestad's Nutrient Flux Density Model 25 An alternative model of nutrition and growth, the nutrient flux density model has been formulated and demonstrated by Ingestad (Figure 4). The basic premise of the model is that it is the rate of nutrient supply to the roots or relative addition rate (RA) (amount of nutrient added per unit of time and unit of nutrient present in the plant) which is the driving variable of growth within the sub-optimum range up to and including the optimum. Since plant growth is exponential with time in the sub-optimum range, a nutrient alone or nutrients in fixed proportions must be supplied in exponentially increasing amounts (relative addition rate RA) corresponding to the exponential growth of the plant (So) (equation 2), and therefore the relative nutrient uptake rate (Ru) will be proportional to RA and Ro (Ingestad and Lund 1986). Ro = CRA (2) where: Ro • l/W(dW/dt) RA = l/W(dM/dt) c ••*» constant of proportionality W - total plant mass M = nutrient concentration t • time 27 Under field conditions the nutrient flux density (amount of nutrients available per unit of time and unit of area) corresponds to RA (Ingestad 1987). The nutrient flux density may be regarded as nutrient flow which enters the plant, similar to energy flow (Ingestad er al. 1981). A constant relative growth rate and constant internal nutrient concentration can only be maintained where the nutrient supply to the roots increases in proportion to the relative growth. The curve (Figure 4) is not continuous like the classical response curve but consists of a sub-optimum range (the straight line below the saturation point) which is related to RA, and a supra-optimum range (the curve to the right of the saturation point) (Ingestad 1982). In the classical response model where a constant amount of nutrients is added per unit of time the amount added becomes uptake restricting. This is due to the fact that the added amounts become less and less sufficient in relation to the requirements of both the internal concentration and relative growth. There are two consequences. Firstly, the deficiency level is overestimated because the classical response curve is the result of changing internal nutrient state. Therefore, plants can be grown having much lower tissue concentrations when steady state nutrition is maintained. Secondly, the potential maximum growth is underestimated using the classical response curve because of insufficient nutrient addition rates to maintain the growth rate. Deficiency symptoms occur during the lag phase when the growth rate of a plant is adjusting from a higher to a 28 lower RA resulting in a decrease in the internal nutrient concentration. The symptoms disappear once a new steady state is formed between RA and Ro resulting in a stable internal nutrient concentration (Ingestad 1982). At the optimum RA the nutrient requirement is saturated and a further increase of RA does not increase the Ro. The external nutrient concentration increases because the RA is not matched by a corresponding increase in the Ru (Ingestad 1982). It is suggested that steady state nutrition is the characteristic situation under natural conditions because growth adjusts to the nutritional resources of the site (Ingestad 1982). Agren (1985) has put forth the nutrient productivity model in which the relative growth rate is proportional to the amount of a nutrient in the plant with the nutrient productivity being the proportionality factor. The nutrient productivity is a constant for a given species under fixed environmental conditions (Agren 1985). Agren (1988) has produced a single formulation which relates growth to the content of several nutrients. The plant growth rate is proportional to the nutrient content minus a given minimum concentration of the nutrient (Equation 3). The proportionality factor, the nutrient productivity, and the minimum concentration are species-specific. Ro s PA{niin(cn|cn|«>pt) - cn,m«.n) (3) = PnH where: P« is nutrient productivity = (l/M)(dW/dt) 29 = (l/NMRo) Re is plant growth rate • dW/dt Cn.nin is the minimum concentration of a nutrient that oust be present before any growth is realized. Cn.opt is the upper concentration of a nutrient above which no further growth response is obtained. cn is nutrient concentration N s (nin(Cn}Cn|ept) ~ Cn,aln) H is the optimum concentration l/N is nutrient efficiency F. Diagnosis of Nutrient Status and Requirements 1. Plant and Soil Analysis The diagnosis of the nutritional status of trees may be performed in several ways through plant analysis or soil analysis. The aim of diagnostic plant analysis is to use some characteristic of the plant which is reflective of plant nutrient status. Plant analysis may involve examination of visual symptoms of deficiencies or toxicities. However, it would be desirable to make a diagnosis before the nutritional problem has manifested itself morphologically. Other methods of plant 30 analysis include chemical analysis of the foliage, buds, phloem, xylem sap, wood, bark, roots, and litter. Diagnostic soil nutrient analysis commonly attempts to use chemical extractants to remove that fraction of nutrients which may be plant available. An advantage of soil analysis is that the nutrient status of the soil could be determined prior to plantation establishment (Mead 1984). However, this method requires the calibration of the extracted nutrient levels with plant nutrition and growth after determining the chemical extracting solution which can give a useful index of nutrient availability. 2. Foliar Analysis a. Background In this study foliar analysis has been used as a diagnostic tool. Several reviews of foliar analysis have been prepared explaining its theory, sampling, interpretation and limitations. These have been by van den Driessche (1974), Lavender (1970), Everard (1973), Walworth and Sumner (1988), Bates (1971), and Ballard and Carter (1986). Pioneering work in foliar analysis was done by Lundegardh (1951, 1947, 1943), Chapman (1941), Thomas (1937, 1945), Thomas and Mack (1941, 1944), Ulrich (1943), Moser (1940) and Scarseth (1943). I 31 b. Physiological and Empirical Basis of Foliar Analysis The aim of diagnostic foliar analysis calibration is to determine the relationship between plant performance and foliar composition, thus enabling the use of the latter as an index of the nutrient status of a plant. It does not reveal anything about why the plant may have a deficiency, sufficiency or toxicity of a nutrient. There is both a physiological and an empirical basis for foliar analysis. Figure 5 presents a somewhat mechanistic model of tree growth based on physical/physiological processes. It illustrates the relationship between plant nutrient status, leaf area and tree growth. The amount of foliage is an important factor in determining the amount of solar radiation intercepted by the canopy. The amount of solar radiation intercepted in turn is an important factor affecting tree growth. Fertilization with a growth-limiting nutrient in a non-closed coniferous stand can be expected to increase growth by increasing needle biomass (leaf size, area, number), thus increasing the photosynthetic capacity (Linder and Rook 1984). Increases in leaf area after fertilization are related to increases in rates of photosynthesis per leaf area and stem wood production (Linder and Rook 1984). This has been found for Scots pine, Douglas-fir and Pinus nigra (Linder and Rook 1984). Vose and Allen (1988) found that leaf area index (LAI) increased up to 60* following H fertilization on two K-deficient sites and that 32 Figure 5. Schematic model of tree growth representation of a detailed (from Landsberg (1986)). mechanistic 33 stemwood production was positively related to LAI. Leyton (1956) detected an increase in the current leaf size which preceded an increase in height growth in the following growing season, induced by changes in soil fertility. Empirical relationships have also been found in terns of correlations between foliar parameters and subsequent tree growth (Table 4). c. Plant Nutrient Diagnosis Using Foliar Analysis i. Total Analysis with Single Nutrients Foliar analysis involves evaluation of the foliage in terms of nutrient concentration, nutrient content, foliar mass, on a foliar area basis or using nutrient ratios. Using combinations of these parameters, several procedures have been used for nutrient diagnosis. The relationship of growth to nutrition based on the classical plant growth curve is the first method which may be used for plant nutrient diagnosis. The first stage of diagnosis involves inferring the existence of one or more nutrient deficiencies or toxicities through the use of visual symptoms, by comparing foliar levels to those plants having superior growth, or by comparing foliar levels to critical levels for the species (applying an existing calibration) or for other species. Since the relationship in part of the deficient zone is quasi-linear, linear regression analysis has sometimes been applied for 34 Table 4. Linear correlations (ra) between growth response and current needle N concentration (NX), N content (Nc) and unit dry weight (Wt) in the two years following fertilization. All stands were N deficient (from Timmer (1979)). Response Response Season NX Nc Wt Species Parameter Period (year) Leader 2 1 0 .67 0, .70 0, .79 Balsam fir Length 2 0 .30 0. .61 0, .62 Shoot 2 1 0 .72 0, .76 0, .77 Balsam fir Length 2 0 .49 0, .74 0. .58 Basal 2 1 0 .69 0, .76 0. .76 Loblolly pine Area 2 0 .36 0, .49 0, .56 Height 2 1 0 .81 0. .86 0, .90 Loblolly pine 2 0 .82 0. .87 0. .88 Basal 3 1 0 .66 0. .85 0. .65 Jack pine Area 2 0 .31 0, .36 0, .69 Volume 5 1 0 .59 0. .66 0. .58 Douglas fir 2 0 .62 0, .81 0. .93 35 diagnostic purposes. This has been extended to the use of multiple regression to infer whether one or more nutrients may be limiting growth. These regression procedures are applied to untreated stands. Those elements with the most significant positive correlation coefficients are considered to be deficient and those with zero and negative correlation coefficient are considered to be sufficient and toxic, respectively. Regression analysis can be problematic if a limiting nutrient is invariant, if the variability of a non-nutritional factor is unaccounted for, or if measured variables lie outside the range where the relationships are linear. Regression analysis has been used by Leyton and Armson (1955) in Scots pine, Leyton (1956, 1957) in Japanese larch, and Prusinkiewicz (1982) in Scots pine. White and Mead (1971) demonstrated the use of multivariate discriminant analysis of foliar nutrients to help distinguish between trees having green and yellow foliage. The second stage in nutrient diagnosis involves demonstration of the deficiency or toxicity. The demonstration of a deficiency or toxicity requires the application of the nutrients in question, through screening trials, and measurement of the subsequent nutrient uptake and growth. If a stand is in the deficient zone one would expect to obtain a response to a wide range of nutrient levels. Regression (linear, multiple or curvilinear) can also be applied to analyse or interpret growth response to nutrient 36 treatments. Regression models have been produced in which foliar nutrient concentrations have been correlated with some function of growth such as height, site index, volume production, mean or periodic increment (Bevege 1984). Interpretation of nutrient growth response data using foliar nutrient concentration and growth alone can be problematic due to the concentration and dilution effects. To overcome these problems a second method, vector analysis as described by Timmer and Stone (1978), simultaneously examines nutrient concentration, nutrient content and foliar mass. The vector analysis method and its interpretation are presented in Figure 6 with additional interpretations by Jarrell and Beverly presented in Table 5. Interpretation of growth response is based upon the direction and extent of the shift of the vector. A third method, the boundary line model, uses data accumulated through field surveys to identify optimum foliar nutrient values (Figure 7). When a plant is close to its optimum nutrient value there often is very little relationship between nutrition and growth (Sumner 1978) which is represented by the boundary line model. In this situation other interacting factors cannot be controlled with the result being the data represented as an array of points. The scatter of points may be due to errors of measurement, variability of the biological material and the overall variation caused by other interacting factors (Webb 37 FOLIAR MASS (g shoot"1) • ELEMENT CONTENT (ug shoot-1) DIRECTION OF SHIFT RESPONSE IN CHANGE IN NEEDLE NUTRIENT NUTRIENT POSSIBLE WEIGHT CONC. CONTENT STATUS DIAGNOSIS A B C D F + + + O O + + ++ + + + + ± DILUTION UNCHANGED DEFICIENCY LUXURY CONSUMPTION EXCESS EXChSS NON-LIMITING NON-LIMITING LIMITING NON-TOXIC TOXIC ANTAGONISTIC Figure 6. Vector method for the interpretation of nutrient-growth response data using nutrient concentration, nutrient content and dry mass of needles (from limner and Stone 1978)). 3a Table 5. General representation of changes in total content, yield and concentration as affected by imposed treatments. An increase is represented by (I), a decrease by (D) and no change by (0) (From Jarrell and Beverly (1981). Case Change in Comments Content Yield Concentration II I I Synergism 2 I 10 3 II D Dilution 4 10 I Synergism 5 ID I Concentration 6 0 0 0 No Response 7 D I D Dilution 8 DO D Antagonism 9 D D I Concentration 10 D D 0 11 D D D Antagonism 39 0 _| , ' ' * - i 0.00 0.02 0.04 0.06 LEAF N/DM Figure 7. An example of a boundary line confining the data representing over 8,000 data points of maize yield versus leaf nitrogen:dry matter (N:DM)(g kg-1) (from Walworth and Sumner (1988) ) . 40 1972). This method sets about consciously varying the controllable growth factors as much as possible by collecting a bank of observations that represent the variability encountered in the real world (Walworth et al. 1986). Therefore, the data actually represent different response curves from single-factor experiments as in Figure 8. A response curve from a single-factor experiement may follow any one of the curves depending on the degree of yield limitation exerted by other factors. Curve fitting may be used to fit a model which describes the boundary line response surface. A point on the boundary line represents the maximum attainable yield at a given foliar concentration under a given set of conditions (Walworth and Sumner 1988). This is not the same as the maximum yield attainable where all growth factors are optimal (Sumner 1978). ii. Total Analysis using Nutrient Balances A number of other methods take nutrient balance into account. The nutrient-element balance concept was introduced by Shear er al. (1946). Sumner (1978) used the boundary line model with nutrient ratios. He then considered a number of ratios simultaneously. By combining the information obtained from each ratio, the order in which the plant requires these nutrients is obtained. Prevot's factorial method uses factorial experiments to 41 Zone of Balanced Zone of X Insufficiency Nutrition X Excess or r Y Excess Y Insufficiency Soil Nutrient (X) Level • or Tissue Nutrient Ratio (X/Y) Figure 8. Diagrammatic representation of crop response to a number of limiting factors (from Walworth and Sumner (1988)). 42 study and calibrate increasing levels of one or more factors while all other conditions are kept constant. From these calibrations one is able to determine the relative proportions of interacting nutrients which would result in balanced nutrition. Kenworthy's balance indices (Walworth and Sumner 1988) are based on foliar optima generated by averaging tissue values of healthy plants gathered from survey data. These values are specific to the stage of growth and position of the sampled foliage. The results from analysis of a sample are compared to the norms and also weighted using coefficients of variation which represent the normal variations of the standard values. This is done using nutrient indices which are calculated as follows. Balance Index = (x/sHlOO + (i - (x/s))(cv) when x < s Balance Index « (x/s)(100 - (1 - (x/s))(cv) when x > s where x = nutrient concentration s » standard (optimum) value cv = coefficient of variation (in percent) of s Nutrients are then ranked in order of requirement with the nutrient having the lowest index being the most required. The Noller-Nielsen Technique attempts to overcome problems of physiological age and nutrient interactions. Four steps are 43 involved. The first step is the production of response curves from nutrient response experiments. With the aid of these curves individual plant samples are corrected back to a standard plant mass. In the second step, standard nutrient values are developed from factorial experiments using the boundary line method to determine optimal concentrations. The plant samples are then compared to the boundary line values and the maximum attainable yield is determined. The most limiting nutrient is the one with the lowest maximum yield. In the third step, boundary line curves for plots of interacting nutrients are used to determine the optimum levels of other nutrients at the existing level of the most-limiting nutrient. The final predicted yield is determined by estimating the yield reduction due to each nutrient. The DRIS method (Diagnostic and Recommendation Integrated System) (Walworth and Sumner 1988) attempts to overcome problems associated with plant age, nutrient interactions, and foliar optima determination. Foliar optima are calculated by averaging nutrient levels from healthy or high yielding plants. The deviations from the mean optimum value are estimated by the coefficients of variation of the high yielding plants. Indices are then calculated for each nutrient using nutrient ratios in the following equations. A index • (f(A/B) + f(A/C) + f(A/D) .+ f(A/M))/2 44 where f(A/B) - (((A/B)/(a/b))-l)(1000/cv) when (A/B) > (a/b) where f(A/B) = (l-((a/b)/(A/B)))(tOOO/cv) when (A/B) < (a/b) A/B is the value of the ratio of the two elements in the tissue under diagnosis a/b is the value of the corresponding norm z is the number of functions cv is the coefficient of variation of the norm The magnitude of each nutrient index represents the relative excess (positive value) or deficiency (negative value) of the nutrient in the tissue. Ingestad (1979) developed a method using nutrient balance. Nitrogen is expressed as 100 and all other nutrients are expressed relative to nitrogen. A set of ratios was developed for macronutrients in hemlock, and for micronutrients in some conifers of the family Pinaceae (Ingestad 1979). iii. Analysis of Separate Fractions Foliar concentrations in themselves do not necessarily indicate anything about the physiological requirements of a nutrient. With wild plants, nutrient levels may serve both a 45 physiological as well as an ecological function in the plant's strategy, as in accumulator plants. Total levels may not necessarily be reflective of the physiological nutrition of the plant because of compartmentation of nutrients or differences in the distribution of nutrients among chemical fractions. Therefore, it would be desirable to measure the physiologically active fraction of a nutrient in order to diagnose the nutrient status of a plant and correlate that nutrient concentration with biochemical and physiological activities. The "functional nutrient requirement" of a plant is "the minimal nutrient concentration which can sustain its metabolic function at rates which do not limit growth" (Ohki 1987). These methods involve isolation and analysis of separate cellular components, analysis of different chemical fractions, enzyme analysis, the measurement of physiological processes, the use of extractants, in situ analysis, and the use of cell cultures. The methods which have been used for Zn and Mn will be discussed. Memon and Yatazawa (1984) isolated different cellular components of Mn accumulator plants using differential centrifugation. The cell wall material, chloroplasts, mitochondria, ribosomes, and vacuolar contents were separated using this method. 46 Enzyme activity has been used as a method to diagnose nutrient status. The activity of the Mn isozyme of superoxide dismutase (Mn-SOD) in pea leaves was directly related to the Mn nutrient levels (del Rio et al. 1978). It was suggested that this isozyme could be an indicator of the biologically active Mn involved in cell metabolism (del Rio er al. 1978). The activity of IAA oxidase was found to increase in cotton with Mn toxicity (Morgan er al. 1966). In bean leaves, Mn toxicity increased the activities of isocitric dehydrogenase and malic enzyme (Anderson and Evans 1956). The activity of ribonuclease (RHAase) was found to be a sensitive, reliable and better index for detecting Zn deficiency in rice and maize than Zn concentration (Dwivedi and Takkar 1974). There was an inverse relationship between Zn supply and RNAase activity. Carbonic anhydrase activity is related to the Zn nutrient level. A shortage in Zn supply to spinach drastically reduced carbonic anhydrase levels (Randall and Bouma 1973). Carbonic anhydrase activity from foliage was used to detect Zn deficiencies in pecan (Snir 1983), and in maize (Gibson and Leece 1981). Rahimi and Schropp (1984) also used carbonic anhydrase activity as an indication of the Zn nutritional status of maize, millet, tobacco, sugar beet and grape. The activity of SOD which was a Cu-Zn isozyme in cotton leaves was used as an indicator of Zn status (Cakmak and Marschner 1987). Water-soluble Zn from foliage has been found to be a suitable indicator of Zn nutritional status. Cakmak and 47 Marschner (1987) found the concentration of water-soluble Zn in leaves to be closely correlated with visual Zn deficiency symptoms and superoxide dismutase in cotton. In orange trees, visual Zn deficiency symptoms in leaves were closely related to the concentration of water-soluble Zn (Cakmak and Marschner 1987). This supports the work of Rahimi and Schropp (1984) who also found that water-soluble Zn was a better indicator of Zn nutritional status than was total Zn. This was due to the direct relationship between water-soluble Zn and the activity of carbonic anhydrase. The possibility of quantifying the levels of nutrients in plants in situ and examining their distribution has been demonstrated using X-ray microanalysis. The first system which has been used is electron probe X-ray microanalysis (EPMA). In this system, electrons excite atoms in a sample, resulting in the production of characteristic X-ray patterns which can be measured. A scanning electron microscope is used in conjunction with the electron microprobe. A description of the instrument and its application in biology has been given by Hall (1979). EPMA has been used by Memon et al. (1980) and Memon and Yatazawa (1982) looking at Mn in Mn-accumulators, by Horiguchi and Morita (1987) who looked at Mn in leaves of barley, and for the study of Zn accumulation in the roots of Betula (Denny and Wilkins (1987), and in the roots of Deschampsia caespitosa (Van Steveninck ec al. 1987). 48 A second instrument which has begun to be used in plant research for in situ anal/sis is the scanning proton mlcroprobe (SPM). A description of the instrument and its use in biological research has been reviewed by Legge and Nazzolini (1980), Legge (1982), Enderer (1982), Legge et al. (1982), Legge (1980), and Legge er al. (1979). This combines a scanning mode with a proton mlcroprobe which utilizes proton-induced X-ray emission (PIXE) (Legge er al. 1979). SPM has enhanced sensitivity compared to EPMA-SEM because the background radiation which is produced (Bremsstrahlung) is orders of magnitude lower (Mazzolini er al. 1985). Consequently, this allows one to detect elements in a sample down to 1 pg g~* (Legge et al. 1979). The SPM instrument could also be used to detect and measure isotopes using nuclear scattering (Legge er al. 1979). The application of the PIXE in plant nutrition studies has been demonstrated by Mazzolini er al. (1985) using wheat seeds, and in the foliage of Eucalyptus obliqua (Mazzolini er al. 1982). The instrument allows one to produce a quantitative elemental analysis of different tissues, as well as producing elemental maps showing the distribution of the elements in the tissue. G. Some Conclusions Hemlock is a species which may be classified as a calcifuge plant because hemlock has in common with this group the following characteristics. These plants favor acid soils and they have ammonium-based nitrogen nutrition. Plants which 49 utilize ammonium-nitrogen can in fact promote acidification of the surrounding soil. Acid soil characteristically has high plant available levels of aluminium and manganese. Hemlock thrives under these acid soil conditions. Hemlock also favors organic rich media which may keep Mn in the reduced state. Tissue nutrient concentrations in a plant are not necessarily reflective of the physiological requirements for that particular nutrient. This is evident for Mn where there is abundant evidence that Mn levels in plants are not reflective of physiological requirements. Nutrients not only have a physiological role but may also play a role in the ecological strategy of a plant. There are three types of ecological strategies of plant nutrition: the accumulator, the excluder and the indicator. Western hemlock has lower foliar Zn levels and higher foliar Mn levels as compared to conifers in other genera in B.C. and the U.S. Pacific Northwest. Whether these differences represent actual different physiological requirements of species, or are an indication of a Zn deficiency and Mn toxicity in hemlock requires some investigation, due to the possibility of increasing productivity through alleviation of these stresses. A review of the literature leads to some conclusions useful in selecting research methods. The classical method (a single application of a fertilizer dosage) was used due to the 50 greater ease in establishing and carrying out the treatments. Diagnosis of foliar data was performed using the vector method. Investigation of the physiological active fractions of Zn and Mn were made using analyses of extractions and of foliar cellular fractions. 51 CHAPTER 3. METHODS AMD MATERIALS A. Site Description 1. Location of Study Areas Fertilizer screening trials were established in five different stands in the Vancouver Forest Region in Coastal British Columbia (Figure 9). Information on the location of the stands is given in Appendix A. Three of the stands are located at the University of British Columbia Research Forest, referred to as sites 1, 2 and 4. The remaining two stands are located at Chipmunk Creek (Chilliwack Provincial Forest) and Mission Tree Farm referred to as sites 3 and 5 respectively. Sites were selected on the basis of their proximity to Vancouver, supporting a uniform stand of trees and consisting of enough trees to support a fertilizer trial. Sites were not selected on the basis of any specific site characteristic. , Sites were classified according to Klinka er al. (1984). Site 1 is located in the Pacific Ranges Drier Maritime Coastal Western Hemlock biogeoclimatic subzone. According to Klinka et al. (1984), the climate is characterized by 57 mm of precipitation in the driest month, warm summers (17.6°C warmest month mean, 33.3°C absolute maximum), mild winters, no month with a mean minimum temperature below 0°C, a mean annual precipitation 52 53 of 1860 no, with 5% as snow, and a range between minimum winter and maximum summer means of 15.2°C. Sites 2, 3, 4, and 5 are located in the Windward Montane Maritime Wetter Coastal Western Hemlock biogeoclimatic subzone. According to Klinka et al. (1984), the characteristic climate in this zone is intermediate between the Windward Submontane Maritime Coastal Western Hemlock zone and the Maritime Forested Mountain Hemlock zone. A climate station is not located in this variant; therefore there are no climate data. 2. Stand Characteristics Sites 1, 2, 3, 4, and 5 were composed of young uneven-aged western hemlock of natural origin. In 1987, the estimated age of the trees ranged from about 10 to 20 years old and they were "free-growing". These stands were characteristic of western hemlock, having clumps of trees with the spaces between clumps being filled in by individual trees. Crown closure had not yet occurred in these stands. Further descriptions on the species composition of these stands is presented in Appendix A. 3. Soil Characteristics Soil profiles of the five sites were described according to the Canadian system of soil classification (Canada Soil Survey Committee 1978). The soils at sites 1 and 3 were classified as Orthic Humo-Ferric Podzols, at site 2 as a Rego Gleysol, at site 54 4 as a Duric Humo-Ferric Podzol, and at site 5 as an Orthic Ferro-Humic Podzol. The chemical characteristics of the soil profiles are presented in Table 6 and other soil characteristics in Appendix A. The methods used for chemical analysis of the soils are described in section G. B. Experimental Design The experiment was carried out using single trees as plots (Viro 1967) in a randomized block design with the blocks acting as replicates. There were ten replicates per treatment at each of the sites. The number, types and year of treatments varied between sites (Table 7). On each site, trees were selected according to the guidelines given by Ballard and Carter (1986). Dominant and codominant trees were selected which were devoid of deformities, insect and diseases, and cone crops. The minimum distance between selected trees was six meters. There was a compromise between having trees close enough to decrease the effect of site variability, but far enough to prevent contamination from adjacent treatments. The trees were identified with plastic tags. Table 6. Chemical characteristics of the soil profiles. SITE HORIZON DEPTH Zn Hn K Ca Mg P Al -cm — 1 1 LFH 3-0 11.8 67 .2 221 718 57 .0 33 1891 1 2 Bf 0-42 1.4 7 . 4 43 62 6 .8 0 2084 1 3 C 42-52 0.9 2 .0 12 48 7 .3 0 2148 2 1 LFH 15-0 67.0 72 .0 295 2566 528 . 0 131 814 2 2 Cg 0-18 7.8 5 .7 129 619 77 .0 52 991 3 1 L(f ) 2-0 2 . 1 2 . 1 74 115 35 .0 0 2052 3 2 Ae 0-5 1.1 9 .3 105 65 16 .0 0 2114 3 3 Bf 5-20 0.5 5 .0 255 16 3 . 0 0 2102 3 4 Bf 20-35 0.3 3 .0 51 8 1 .4 0 2099 3 5 Bf 35-60 0.3 3 .0 0 6 1, .0 0 1894 3 6 Cg 60 + 0.5 6 .0 23 5 0 . .8 43 1710 4 L LFH 20-0 39 .0 129 .0 404 1782 224. .0 51 1049 4 2 Ae 0-10 0.8 3 .2 37 96 3 . ,0 0 2022 4 3 Bf 10-46 1.9 9 .7 0 26 5 . ,6 0 2079 4 4 BCc 46-72 2.2 20 .0 0 47 6 . ,4 2 2039 4 5 C 72 + 0.5 0 .5 48 24 2 . ,2 0 2075 5 1 LFH 25-0 30.3 51 .7 212 2471 234. 0 26 1412 5 2 Bhf 3-6 3.6 2 . 4 86 369 52 . 0 6 1869 5 3 Bhf 6-18 0.6 0 .4 0 45 8 . 3 0 2123 5 4 Bf 18-27 0.7 0 .5 45 53 10 . 0 0 2141 5 5 Bf 27-35 1.1 1 .2 78 80 17. 0 0 2054 5 6 C 35 + 0.3 0 . 4 57 25 2. 3 0 2123 SITE HORIZON DEPTH Fe Cu B Extr . N Total CEC PH g"1 -1 _ meq 100 g" 1 HaO 1 1 LFH 3-0 249 1. 5 0 0.006 0.062 6 . .0 3 .9 t 2 Bf 0-42 48 1. 1 0 0.003 0.195 12 . .1 4 .9 1 3 C 42-52 204 0. 9 0 0.005 0.213 21, .7 4 .5 2 1 LFH 15-0 174 1. 6 0 0.015 0.907 16 . .0 3 .6 2 2 Cg 0-18 157 1. 0 0 0.003 0.218 14, .0 3 .7 3 1 L(f) 2-0 381 0 . 4 0.2 0.003 0.076 20 , .3 3 .8 .8 3 2 Ae 0-5 106 1. ,0 0 .08 0.006 0.039 12 .5 4 3 3 Bf 5-20 106 0. 7 0 .06 0.003 0.076 9 . .9 4 .8 3 4 Bf 20-35 51 0 . 7 0.05 0.002 0.057 4, . 7 4 .9 3 5 Bf 35-60 71 0.5 0.04 0.003 0.028 1, .0 4 . 8 3 6 Cg 60 + 40 0 . ,4 0.03 0.004 0.016 0 4 .8 4 1 LFH 20-0 238 2 . 2 0 0.016 0.709 12 , . 1 3 .6 4 2 Ae 0-10 59 1. .1 0 0.002 0.076 5 , .1 5 .0 4 3 Bf 10-46 125 1. 1 0.08 0.002 0.04 0 . .8 4 . 7 4 4 BCc 46-72 130 0 . 8 0.07 0.004 0.256 9 . .5 4 .4 4 5 C 72 + 68 1. 0 0.03. 0.005 0.072 1. .9 4 .4 5 1 LFH 25-0 223 2 . .2 0 0.008 0.755 6 , .9 4 .2 5 2 Bhf 3-6 135 2 . ,2 0 .08 0.007 0.074 24, .0 4 . 3 5 3 Bhf 6-18 44 0. .8 0 .03 0.003 0.233 21, .2 4 4 . 6 5 4 Bf 18-2 7 67 1. .0 0.02 0.005 0.317 13 , .0 .6 5 S Bf 27-35 88 1. .0 0 .001 0.005 0.259 15 , .1 4 .5 5 6 C 35 + 20 1. .2 0 . 1 0.001 0.09 5 , .6 4 . 9 pH 3.4 4.4 4.1 3.2 3.1 56 Table 7. Treatment-levels used in the fertilizer trials. Sites 1, 2, and 3 Application Method Soil Number 1 2 3 9 10 12 ZnSO* ZnSO* MnSO* NaaSO* NaaSO* Control Rate 10 kg Zn ha"1 50 kg Zn ha-1 200 kg Mn ha~v 5 kg S ha~* 25 kg S ha"* Foliar 4 5 6 7 8 tl ZnSO* ZnSO* MnSO* HaaSO*. NaaSOA Control 360 mg Zn L~x 3600 mg Zn L~x 2730 mg Mn L~x 177 mg S L-1 1770 mg S L~x Demin HaO + Surfactant Table 7 (continued) Sites 4 and 5 Soil 1 2 3 4 5 6 7 8 9 10 11 12 Foliar 13 14 15 16 17 18 19 20 21 22 24 ZnSO* 50 kg Zn ha~ X ZnSO* 100 kg Zn ha~ 1. ZnSO* 200 kg Zn ha~ X MnSOd. 200 kg Mn ha~ X MnS0A 400 kg Mn ha~ X MnSO* 600 kg Mn ha~ X S 25 kg S ha~* S 49 kg S ha"1 S 98 kg S ha"1 s 118 kg S ha"1 s 235 kg S ha"4 s 352 kg S ha~* ZnSO* 360 mg Zn L~* ZnSO* 1800 mg Zn L~* ZnSO^ 2700 mg Zn L_1 MnSO* 2730 mg Mn L~l MDSOA 4095 mg Mn L~* NaaSO* 177 mg S L~x NaaSO* 883 mg S L-«-HaaSOA 1325 mg S NaaSOA 1605 mg S L~x NaaSOA 2408 mg S L"1 Control Demin HaO + Surfactant Table 7 (concluded) Soil 23 "Complete -Zn -Mn" Urea 100 kg N ha_t Triple Super Phosphate 150 kg P ha"1 KSSOA 22 kg K ha"1 MgSO* 18 kg Mg ha"1 CaSO* 11 kg Ca ha"1 Degra-sul 22 kg S ha"1 CUSOA 4.4 kg Cu ha~ Solubor 0.9 kg B ha"1 FeSOA 11 kg Fe ha"1 25 Control 59 C. Fertilizer Treatments Fertilization can be used as a tool to diagnose the nutrient status of a stand. The purpose is to increase the supply of the nutrient of interest. Then, using the dose-response curve and vector analysis, one can evaluate the nutrient status of the stand. The fertilizers used were zinc sulphate, manganese sulphate, sodium sulphate, elemental sulphur (as Degra-sul), urea, triple super phosphate, potassium sulphate, calcium sulphate, magnesium sulphate, iron sulphate, copper sulphate, and boron (as Solubor). The specific treatments used for each site are outlined in Table 7. There were a total of 120 trees in the trials on each of sites 1, 2, and 3, and 250 trees on each of sites 4 and 5. Fertilizer was applied in solid form to the soil and in solution as a spray to the foliage. The soil and foliar treatments were applied at different times. The soil treatments were applied in May, and the foliar treatments were applied at the beginning of July when there was a large amount of new growth. For sites 1, 2 and 3, the trials were established in 1985. For sites 4, and 5 the trials were established in 1986. On sites 1, 2, and 3, sodium sulphate was used as the sulphur source for the foliar and soil treatments. On sites 4 and 5, elemental sulphur was used as the sulphur source for the soil treatments 60 and sodium sulphate for the foliar treatments. The "complete -Zn-Mn" treatment was applied only to soils, and only at sites 4 and 5 . The fertilizer solutions were prepared using deraineralized water. A commercial detergent (trade name 'Joy') at a concentration of 0.5% by volume was added to the fertilizer solution as a surfactant to enhance nutrient absorption by the foliage. The fertilizer solutions were applied using a backpack plastic sprayer. The trees were sprayed until the solution began to drip from the canopy (approximately one litre per tree). In this way, if there were canopy size differences between trees, each unit area of foliage of each tree will have had the same rate of nutrient application. D. Field Sampling The sampling procedure was carried out according to the guidelines given by Ballard and Carter (1986). Foliage and shoot samples were collected from September 15-December 15 from sites 1 and 2 in 1985, from sites 1, 2, 3, 4, and 5 in 1986, and from sites 4 and 5 in 1987. For sites 4 and 5 only replicates 1-5 were sampled in 1986. All replicates were sampled in 1987. For all sites and sampling years both the current and previous year's foliage and shoot samples were collected using hand and pole pruners. Foliage was collected from the upper one-half to one-quarter of the crown but below the third whorl. A shoot sample 61 refers to the tip growth of a branch formed in the current year. Three shoot samples per tree were taken from the third whorl for growth analysis. For site 3, shoot samples were taken for the current year (1986) and the previous two years. For sites 4 and 5 in 1987, branch samples were collected from the third and fourth whorls from the top consisting of the previous three years of growth for replicates 6-10. This allowed for analysis of shoot increment for 1986 at whorl three for all 10 replicates. Foliage and branch samples were placed in plastic bags, transported to the laboratory, and stored in a cold room until further processing. Height increment measurements were taken in the fall of 1986 on sites 1, 2, and 3 for the previous three years of growth, and in 1987 on sites 4 and 5 also for the previous three years of growth. Height increment measurements were made to the nearest 0.5 cm. At the time of sampling trees of other species were also sampled. One codominant or dominant tree of other species occurring adjacent to the control hemlock tree per block was sampled. Soil samples were collected from sites 1, 3, 4, and 5 in the fall of the year in which the fertilizer trial was established. Samples were collected from around each control hemlock tree at a distance of 30 cm from the base of the stem. Up to three samples were collected at intervals of 120° both of the forest floor and of the mineral soil down to 15 cm. These are identified as (T) for forest floor and (B) for mineral soil. Mineral soils were composited in the field. 62 A soil pit was excavated at each of the sites for a description of the profiles. In addition, soil samples were collected from HHr.h horizon for ohHmi.cal. anal/His. Data from the soil pit were used only for descriptive purposes; no statistical interpretations were made from the soil pit data. Foliage samples were separated into current and previous-year's portions. Shoot lengths of the current and the previous year's growth were measured to the nearest mm. Foliage was oven-dried at 70°C for 24 hours in paper bags. Growth measurements were made on the samples used for the shoot increment measurements. These were foliar mass per shoot to the nearest milligram, and number of needles per shoot. From these values the mass per needle, the needle mass per cm of shoot and the number of needles per cm of shoot were calculated. In addition, for the samples from the control western hemlock trees and samples of trees of other species, the mass of needles per 100 needles was also determined to the nearest milligram. Dry foliage samples to be used for chemical analysis were ground in a Braun type KSM-2 coffee grinder and stored in air-tight plastic containers. The shoot samples gave a data base of 2,580 samples for both first and second year. Approximately 490 samples were used to measure first year nutrient response, and 860 in the second 63 year. There were approximately 860 height increment values for each of three years for the entire experiment. E. Chemical Analysis The wet digestion method of Parkinson and Allen (1975), slightly modified by Ballard (1981) was used for the determination of total N, P, K, Ca, Mg, Mn, Zn, and Al (Ballard and Carter 1986). N and P were measured on the original digest by colorimetric analysis for N (phenol-hypochlorite method) and P (unreduced vanadomolybdate complex), by the Technicon Autoanalyzer II. The elements K, Ca, Mg, Mn, Zn, and Al were measured using atomic absorption spectrophotometry (AA) (Perkin Elmer 306). For foliage from 1986 and 1987, the elements P, K, Ca, Mg, Mn, Zn, and.Al were measured using Inductively Coupled Plasma Emission Spectroscopy (ICP) (Jarrell Ash AtomComp Series 1100) on the original digests. Details of the method are described in Appendix Bl. The elements Fe and Cu were determined using a nitric acid digestion followed by atomic absorption spectrophotometry (Ballard and Carter 1986) (Appendix B2). Although there was a good agreement between the standard foliage samples using AA and ICP for Fe analysis using the Parkinson and Allen digest, there was poor agreement for the hemlock samples (Appendix C). Copper was found to be below the detection limits of the ICP using the Parkinson and Allen digests. The standards were used to construct equations in order to convert Zn and Mn concentrations measured on the AA in the first year (1985) to 64 equivalent values for the ICP (Appendix D). The equations were used in the retranslocation study to compare first year values measured on the AA to foliar values from the second year (1986) measured on the ICP. A comparison was made between AA and ICP for the National Bureau of Standard samples in the percentage recovery of Ca, Mg, K, Mn, Zn, and Al from foliage. This information is presented in Appendix E. Boron was determined by dry ashing, followed by colorimetric analysis by the azomethine H method of Gaines and Mitchell (1979). Total sulphur was determined using a Fisher Model 475 Sulphur Analyzer using the procedure described by Guthrie and Lowe (1984). Sulphate-sulphur was extracted from the foliage according to the procedure outlined in Appendix B3 and determined according to the method described by Kowalenko and Lowe (1972) (Appendix B3). The idea that only a portion of the total level of an element may be physiologically active led to the need to determine active iron, and water-extractable zinc and manganese. The concept of a metabolically active fraction of iron was investigated by Oserkowsky (1933). His procedure for the determination of active iron, slightly modified by Ballard (1981), is described in Appendix B4. The physiologically available zinc was determined using a modified procedure of 65 Cakmak and Marschner (1987) described in Appendix B5. Extractable manganese was determined using a modified water extraction method of Memon and Yatazawa (1982) (Appendix B6). The cellular fractions in foliage were separated using the method of Memon and Yatazawa (1984) slightly modified by this author described in Appendix F. Total zinc and manganese were determined in the different fractions using the Parkinson and Allen digestion. Three replicates were done for each sample. One experiment compared zinc and manganese levels in different fractions from the high foliar zinc treatment and the high soil manganese treatment. These samples were from current year's foliage in the first season following fertilization (1986) from site 5. The second experiment compared zinc and manganese levels in the foliage from different species using the current year's foliage of 1987 from site 5. F. Scanning Electron Mlcroprobe In addition to comparing the nutrition of species using total nutrient levels, extractable levels and cellular fractions, an attempt was made to measure the nutrients in the foliage of different species in situ. This was done using electron probe microanalyzer (EPMA) with scanning electron microscopy (SEM). The tissue was fixed and prepared partly in the field and the laboratory according to the method described in Appendix G. The SEM-EPMA in the Department of Metallurgy at the University of 66 British Columbia was used. However, the resolution of EPMA is not fine enough to measure micronutrients in the range of ug s~%. Alternatively, an attempt was made to have the analysis done on a scanning proton mlcroprobe (SPM); however, the price of the analysis was cost-prohibitive. G. Soil Sample Preparation and Analysis The mineral soil and forest floor samples were air-dried at room temperature (22*C). The soil samples were sieved through a 2.0-mm stainless steel mesh sieve. Forest floor samples were ground in a Waring blender to pass a 20-mesh sieve. The samples were analyzed for pH, cation exchange capacity, total N (TH), extractable NH*~ (EXTN), P, K, Ca, Mg, Mn, Zn, Cu, and Fe. Soil pH was measured in water and 0.01 M CaCls, using a glass electrode pH meter. The soil to solution dilution ratios were 1:2 for mineral soils and 1:8 for organic soils. Cation exchange capacity was determined using the sodium chloride method. This is an unbuffered solution enabling evaluation of the cation exchange capacity of the soil at its inherent pH. Total N was determined using semi-micro KJeldahl digestion followed by colorimetric determination using the Autoanalyzer. Available MHA* was assayed using an extraction in 2% KaSO* followed by colorimetric determination using the Autoanalyzer. Available P, K, Ca, Mg, Mn, Zn, Cu, and Fe were 67 extracted using the Mehlich 3 soil test extractant (Mehlich 1984) as modified by Ballard (Appendix H). The extractants were analyzed by ICP. H. Measurement of Fertilizer Response Response to fertilization was measured using various growth parameters: height increment ratio, shoot length increment ratio, foliar mass per shoot and foliar nutrient concentration. Where there was a significant difference in foliar mass: foliar nutrient concentration, nutrient content per shoot and foliar mass per shoot were evaluated together as a measure of growth response IINI'IIK the v«i:l:or; method off TimniKr and Stone (1.978) (Figure 6). Results of the "complete-Zn-Mn" treatment were presented using vector analysis on a relative basis. This permits comparison of various elements together on one graph. The ascending order of elements along a relative unit weight line indicates the degree of deficiency for these elements (Timmer and Morrow 1984). Otherwise nutrient concentration was used alone. Interpretation of foliar nutrient concentrations was done using the summary of Ballard and Carter (1986) presented in Appendix I. Growth and nutrient responses were evaluated in terras of the statistical significance of the treatments from their respective controls. 68 Relative shoot increment and height increment were also used to evaluate growth response using the pretreatment increment method of Ballard and Majid (1985). The use of pretreatment increment allows adjustment for site as well as stand structure differences. Shoot growth as well as height growth response were expressed as the ratio of post-fertilization to pre-fertilization shoot and height increment. Af/Bf compared with Ac/Be where: A • increment after fertilization B = increment before fertilization f - fertilized c = control The ratio Af/Bf is an index of fertilizer response as well as environmental effects, whereas Ac/Be is an index of only environmental influence. Their difference provides a measurement of solely the treatment effect (Ballard and Majid 1985). Shoot length increment may sometimes be an indicator of future volume growth. Barker (1978) found a correlation (r=0.95) of the difference in shoot increment (between the fertilized and control in the previous year) with the difference in volume increment (between the fertilized and the control) in the current year. 69 I. Statistical Analysis All statistical tests for significance were performed using both parametric and non-parametric methods. There were three groups of data subjected to statistical tests. Because each fertilizer treatment had its own control, the null hypothesis of no treatment effect could be evaluated by parametric two-sample t-tests. If the differences between the variances were more than three-fold, the results were checked using the non-parametric Mann-Whitney test. For the experiment involving comparison of foliar nutrient concentrations between species, the null hypothesis which was tested was that there were no differences in foliar nutrient concentrations between species. The null hypotheses was tested by a one-way analysis of variance. Differences between means were analyzed using the parametric Tukey multiple comparison test. The Tukey test allows for the unbalanced condition and is generally preferred to the other multiple range tests (Wilkinson 1988). If the variances were not homogeneous, as determined by Bartlett's test, the significance of difference between means was checked using the Kruskal-Wallis test as a non-parametric analysis of variance, and the Mann-Whitney test as a multiple comparison test. The probability table used for the Mann-Whitney test was adjusted using the Bonferroni procedure (Wilkinson 1988) to take into account the comparison of more than one pair of 70 means. Without such an adjustment, there would be increased probability of making a type I error when using a two-sample test for more than two means for reasons described by Zar (1984). The data examining retranslocation in the foliage were analyzed using the parametric paired t-test and the non-parametric equivalent test, the Wilcoxon signed rank test. Relationships between nutrients, between nutrients and growth, and between growth parameters were investigated using curvilinear regression methods. All t-tests, ANOVAs and regressions were evaluated at the 5X level of significance. 71 CHAPTER 4. RESULTS A. COMPARATIVE NUTRITION A comparison of nutrition was made among species occurring in the same stands (Table 8) to determine whether the nutritional characteristics of hemlock are species- or site-related. 1. Total Levels Hemlock tends to have lower foliar Zn concentrations compared with Douglas-fir, amabilis fir and white pine. But it has higher foliar manganese concentrations compared to all the other species examined. 2. Extractable Zn and Mn Although not examined in this study, it is inferred that substantially different concentrations of extractable nutrients in different species on the same site may be indicative of different physiological requirements. The water-soluble fraction of Zn has been shown to represent the physiologically active component by Rahimi and Shropp (1984) and Cakmak and Marschner (1987). Water-soluble Mn has been shown by Memon et al. (1980) to exclude the fraction of Mn bound in the cell wall and the latter may be physiologically inactive. 72 Table 8. Total foliar Zn and Mn, water-soluble Mn (AMN), and active Zn (AZN) for different species in the same stands. Within a column means with different letters are significantly different at the 5% level using ANOVA. Values are nutrient mass/dry matter in ug g-1-. SHE 1 1986 MEAN SPECIES Douglas-fir Amabilis fir Hemlock ZN 22 .3a 17.2a 9 . 7b MN 473a 571a 1139b AZN 19 ,0a 11.5b 8.8b AMN 755a 842ab 1218b SITE 4-1987 MEAN SPECIES ZN MN Douglas-fir 17.2a 515a Amabilis fir 16.0 675a Hemlock 11.7b 1545b SITE 2-1986 MEAN SPECIES Red cedar White pine Hemlock SITE 3-1986 MEAN ZN 12.6a 29 .Ob 11.1a MN 246a 375a 1932b AZN 4.3a 20.7b 12 . lc AMN 117a 439a 1948b Site 5-1987 MEAN SPECIES ZN MN Douglas-fir 14.9 265a Amabilis fir 17.2a 415b Yellow cedar 11.9b 23a Hemlock 11.8b 1243c SPECIES ZN MN Amabilis fir Hemlock 18.3a 11.0b 916a 1505b SITE 4-1986 MEAN SPECIES ZN MN Douglas-fir 20.0a 713a Amabilis fir 19.1a 735a Hemlock 8.7b 1428b SITE 5-1986 MEAN SPECIES Douglas-fir Amabilis fir Hemlock ZN 18 . 0a 10.3b 11.4b MN 472a 220b 1308c 73 It was hypothesized that although different species may have different total foliar levels of a nutrient, the physiologically active fraction may be similar. Therefore, to examine this hypothesis the active Zn and water-soluble levels of Mn, and total foliar levels of Zn and Mn were compared among species on sites 1 and 2 (Table 8). On site 1, Douglas-fir and amabilis fir have higher total Zn than hemlock. Douglas-fir has higher active Zn than hemlock, but amabilis fir has similar levels of active Zn to hemlock. On site 2, white pine had higher total and active Zn than hemlock, which in turn had higher levels than red cedar. On site 1, hemlock had higher total and water-soluble Mn levels as compared to Douglas-fir and amabilis fir. On site 2 hemlock had higher total and water-soluble Mn concentrations than white pine and red cedar. 3. Cellular Fractions of Foliage Levels of Zn and Mn were compared between control and treated samples, and between species. The data appear in Appendix J. It was hypothesized that although different species may have different total foliar levels of Zn and Mn the levels in the different cellular fractions which represent different physiological processes may be similar. The fractions identified by Memon (1984) were A, corresponding to cell wall and debris; B, chloroplasts; C, mitochondria; D, ribosomes; and E, vacuoles. In the present study, fractions A and B corresponded to Memon's A and B, C contained chloroplasts, D corresponded to the 74 mitochondrial fraction, and E contained the ribosomal and vacuolar fractions. For Zn, the cell fractions from the high Zn treatment had higher levels of Zn than the control fractions (Figure 10). The situation was similar for Mn in that for all fractions the Mn treatment had the higher level of Mn compared to the control (Figure 11). In comparing Zn and Mn treatments accumulation occurred in two different fractions. Zn tended to accumulate in fraction D and Mn tended to accumulate in fraction E. In a comparison of unfertilized trees, there was a difference among species for Mn concentrations in cellular fractions (Figure 12). Hemlock had higher Mn levels in fractions A, B, C, D and E. Yellow cedar had the lowest Mn levels of any species in fractions A, B, C, D and E. There was a predominance for Zn to accumulate in fraction D for all species (Figure 13). Douglas-fir had the highest Zn concentration in the D fraction compared to the other species. 75 Figure 10. Zinc concentrations in different cellular fractions from the current year's foliage (1986) of the control treatment and high foliar Zn treatment from site 5 in the first year of treatment. Fraction A = cell wall and debris, B + C = chloroplasts, D = mitochondria, E = ribosomes and vacuolar contents. 76 3000 Manganese us s--Sample 1 I Control 600 kg Mn ha"1-Figure 11. Manganese concentrations in different cellular fractions from the current year's foliage (1986) of the control treatment and high soil Mn treatment from site 5 in the first year of treatment. Fraction A = cell wall and debris, B + C = chloroplasts, D = mitochondria, E = ribosomes and vacuolar contents. 77 Manganese,ug g_i 1400 | :  1200 -1000 -800 -600 - I" 400 -ABODE Fractions Species I 1 Hemlock •! Douglas-fir CZ! Amabilis fir ZH Yellow cedar Figure 12. Manganese concentrations in different cellular fractions from the current year's foliage in 1987 of unfertilized trees of different species on site 5. Fraction A = cell wall and debris, B + C = chloroplasts, D = mitochondria, E = ribosomes and vacuolar contents. 78 120 Zinc Ms s-i 100 -I J Hemlock 8peciea I Douglas-fir Mill Amabilis fir Yellow cedar Figure 13. Zinc concentrations in different cellular fractions from the current year's foliage in 1987 of unfertilized trees of different species on site 5. Fraction A = cell wall and debris, B + C = chloroplasts, D = mitochondria, E = ribosomes and vacuolar contents. 79 In fractions A, B, and C hemlock tended to have lower levels than the other species. In fraction E, hemlock had lower Zn than Douglas-fir and amabilis fir. In all fractions Douglas-fir tended to have higher Zn concentrations than hemlock. B. Fertilization Experiments 1. Nutrient and Growth Responses Conventionally vector analysis has been applied to species with determinate shoot growth and has been expressed as mass per fixed number of needles. For hemlock, the question has arisen whether foliar mass should be expressed on the basis of a fixed number of needles or on a per shoot basis. Hemlock has both determinate and indeterminate shoot growth, hence its species name heterophylla, hetero meaning different, and phylla meaning leaves (Harlow and Harrar 1958). The distal needles on a 1-year-old shoot are shorter than the proximal needles (Owen and Holder 1973). The proximal needles are the result of determinate shoot growth. These needles were formed in the bud the previous year, and therefore their number would depend upon the environmental conditions of the previous year, but their mass would reflect the current year's environment. The needles formed at the distal part of the shoot are the result of indeterminate shoot growth which means that they were initiated and elongated in the same 80 year. Therefore, both their number and mass would reflect the current year's environmental conditions. It appears that foliar mass per shoot would be an appropriate parameter in which to express growth for two reasons. Firstly, foliar mass per shoot is a function of both number of needles per shoot and mass per needle. Therefore, changes in either of the two components would be reflected in foliar mass per shoot. Secondly, from regression analysis there were significant relationships between current height increment and foliar mass per shoot (Figure 14 and Table 9). The relationship between foliar mass per shoot and current height increment was positive and linear, but in some cases the relationship was curvilinear. The curvilinear relationship indicates that a maximum point was reached beyond which an increase in foliar mass per shoot was associated with a reduction in height increment. In cases where the change in foliar mass per shoot and foliar concentration were determined to be significantly different from the controls, the response was assessed using vector analysis. Otherwise, nutrient concentration was used alone. The nutrient and growth data for the fertilization trials for all sites and years are presented in Appendix L. 81 Figure 14. Scatter plots of the foliar mass per shoot for site 1 second years (1986) (B). height increment versus the in the first (1985) (A) and 82 Table 9. Equations and correlation coefficients (Ra) for the height increment (y) - foliar mass per shoot (x) relationships for each site. Refer to Appendix K for additional scatter plots. 1 1986 y = 171.Ox - 117.Ox* 0.59 2 1986 y « 291.Ox - 440.0xa 0.38 K.l 3 1986 y = 154.Ox - 151.0xa 0.32 K.2 4 1986 y » 160.Ox - 71.0xa 0.55 K.3 4 1987 y • 16.0 + 163.Ox - 82.0xa 0.49 K.4 5 1986 y = 204.Ox - 143.0xa 0.43 K.5 5 1987 y » 206.Ox - 124.0xa 0.49 K.6 83 a. Zinc i. Foliar Zinc Treatments In the first growing season following foliar zinc applications, an increase in uptake of foliar zinc occurred. This was true for all sites and all levels of application (Table 10). In the second growing season following foliar zinc applications, foliar zinc levels were still elevated on sites 1 and 2 from treatment 5 (3600 mg Zn L~M, on site 3 from treatment 4 (360 mg Zn L-1), and on site 5 from treatment 15 (2700 mg Zn L-M (Table 10). On site 2 in the first year there was an increase in foliar mass and foliar Zn from treatment 4 (360 mg Zn L-*-), which implies a growth response (Table 10 and Figure 15A) (shift in C direction). Conversely a reduction in mass and increase in foliar Zn from treatment 5 (3600 mg Zn L~x) indicate a Zn toxicity (Figure 15A) (shift in E direction). On site 3 there was an increase in foliar mass and foliar Zn after the second year, from treatment 4 (360 mg Zn L-1), indicating a growth response (Figure 15B) (shift in C direction). On site 5 in the first year, there was a decrease in foliar mass and increase in foliar Zn from treatment 13 (360 mg Zn L-i), suggesting a Zn toxicity (Figure 15C) (shift in E direction). 84 Table 10. Foliar zinc (ug g-1) concentration response and foliar mass per shoot (g) growth response to foliar applications of zinc in the first and second years following treatments. Values in parenthese are the standard deviation. An (*) indicates a significant difference and n.s.d. indicates no significant difference at the 5% level between a treatment and its respective sulfur control . Sits 1 Site 3 - • First Tear Second Tear Second Tear Treataent Za Kaas Za Haaa Treataeat Zn Haaa —us *"*•- —t— —ft I"*- —s— —us »-*•• —5— 360 i; Zn [,->• 38.1 0.18 13.4 0.264 360 as Za l"-21.3 0.233 (46.3) (0.154) (2.2) (0.193) (2.4) (0.125) 177 >; S (.-"• 177 as S L-» 10.5 ' 0.202 11.3 0.202 14.4 0.171 (3.*) (0.064) (3.3) (0.117) C3.7) (0.039) * a. a . d. a.a.d. a.a.d. * • 3600 »c Za !.-«• 3600 as Zn f» 19.1 0.193 1*9.7 0.241 15.2 0.206 (3.3) (0.081) (46.S) 1770 at S L-1 (0.154) (5.2) (0.149) 1770 as S L-* 17.4 0.172 7.9 0 .208 11.1 0.140 (3.4) (0.103) /' (2.1) (0.076) (3.5) (0.036) a.a.d. a.a.d. a .s . d . * a.a.d. Table 10 (contiaued). Site 2 First Tear Second Tear Treataent Za Haaa Zn Haaa —vi *"*— —t— —us «""•- ' ~t-360 as Za t"» 38. 3 0.153 11.5 0.133 (21.3) (0.034) (5.6) (0.063) 177 at S L-* 6.7 0.107 8.8 0.123 (2.3) (0.019) (3.4) (0.039) • * o.a.d. a.a.d. 3600 at Zn L~* 73.0 0.077 19.6 0.131 (99.0) (0.049) (3.8) (0.092) 1770 at S L-» 3.3 0.118 11.4 0.163 (2. 8) (0.042) (2.7) (0.113) • • * a.a.d. Table 10 (concluded) Site 4 First Year Treatment Zn Kass —VI 8"1 --% — 360 mg Zn L_l 18.3 0.263 (6.9) (0.156) 177 mg S L"1 8.2 0.216 (3.3) (0.231) : * n.s.d. 1800 mg Zn L"1 26.2 0.299 (10.4) (0.082) 883 mg S L"1 9.4 0.267 (3.1) (0.071) * n.s.d. 2700 mg Zn L~l 48.6 0.171 (25.1) (0.136) 1325 mg S L"1 6.4 0.136 (2.1) (0.063) * n.s.d. Second Year Zn "Pg g_l-10.2 (2.6) 8.5 (3.0) n.s.d. 8.3 (2.6) 8.5 (3.1) n.s.d. 9.4 (3.0) 9.7 (3.5) n.s.d. Mass — g — 0.421 (0.340) 0.327 (0.174) n.s.d. 0.371 (0 .266) 0.459 (0.277) n.s.d. 0 .368 (0.293) 0.515 (0.335) n.s.d. Site 5 First Year Second Year Treatment Zn Haas Zn Hasa — Mg g" 1 "g — —vs g~1-- ~g~ 360 mg Zn L-t 79.3 0.184 11.9 0.306 (30.1) (0.078) (2.9) (0.152) 177 mg S L~l 11.2 0.291 10.5 0.278 (1.9) (0.052) (4.6) (0.110) • * n . s". d . n.s.d 1800 mg Zn L-1 100.9 0.173 15 .4 0 .292 (31.8) (0.078) (4.9) (0.104) 883 mg S L-1 10.1 0.198 12.5 0.301 (3.3) (0.071) (5.1) (0.151) * n.s.d. n.s.d. n.s.d. 2700 mg Zn L~l 132.3 0.171 16.5 0 .314 (30.5) (0 .093) (4.5) (0.170) 1325 mg S L-1 10.5 0.174 12.4 0 .308 (1.9) (0.052) (3.5) (0.165) 86 Foliar Mass 100 0.1 0.2 4 6 Zinc Content 0.1S Foliar Mass Z I n c C o n c e n t r a t I o n 140 120 100 -J.25 Foliar Mass 0.15 0.2 10 15 Zinc Content Figure 15. Vector diagrams of growth response to foliar applied zinc. (A) First year in 1985 on site 2, (B) second year in 1986 on site 3, and (C) first year in 1986 on site 5. The x-axis is pg Zn per shoot, the y-axis is ug Zn g-t and the dashed line is foliar mass per shoot in grams. The arrow indicates the direction of the response. Treatments are in mg Zn L-1-. 87 There was no toxicity found with greater applications of foliar Zn. There were no significant effects of Zn application on foliar mass per shoot in the first year on site 1 from treatment 4 (360 mg Zn L~M, on site 4 from treatments 13 (360 mg Zn L~M, 14 (1800 mg Zn L-M, and IS (2700 mg Zn L-*-), and site 5 from treatments 14 (1800 mg Zn L~M and 15 (2700 mg Zn L~"M but increased uptake of Zn occurred, indicating luxury consumption on those sites. ii. Soil Zinc Treatments A different pattern of response was found for the zinc soil treatments. No response occurred in the first growing season following fertilization on all sites. However, in the second growing season, nutrient concentration response occurred from treatments 1 (10 kg Zn ha~x) and 2 (50 kg Zn ha~x) on site 1, treatment 2 (50 kg Zn ha""M on site 3, and from treatment 3 (200 kg Zn ha~l) on site 5 (Table 11). Since there were no incidents of significant increases in foliar mass per shoot, these results indicate luxury consumption of Zn on these sites. On sites 4 a positive growth response occurred in the second year from treatment 3 (200 kg Zn ha~*x) (Figure 16) (shift in C direction). 88 Table 11. Foliar zinc (pg g-1) concentration response and foliar mass per shoot (g) growth response to soil applications of zinc in the second year following treatment. Values in parenthese are the standard deviation. An (*) indicates a significant difference and n.s.d. indicates no significant difference.at the 5% level between a treatment and its respective sulfur control. Site 1 Treatment Zn Mass —s-3- -~s— 10 kg Zn ha"1 13.3 0.248 (3.9) (0.206) 5 kg S ha-1 9.9 0.213 : (2.4) (0.131) * n.s.d. 50 kg Zn ha"1 17.2 0.221 (10.3) (0.120) 25 kg S ha"1 11.4 0.343 (1.5) (0.231) * n.s.d. Site 3 50 kg Zn ha"1 17.7 0.303 (10.0) (0.248) 25 kg S ha"1 12.8 0.303 (4.3) (0.360) Site 4 Treatment Zn Mass -ug g_1- —S— 200 kg Zn ha"1 11.2 0.364 (2.7) (0.195) 98 kg S ha"1 6.4 0.199 (2.3) (0.084) Site 5 200 kg Zn ha"1 15.2 0.258 (5.7) (0.212) 98 kg S ha-1 8.8 0.272 (1.8) (0.108) * n.s.d. 89 Figure 16. Vector diagram of the second year growth response in 1987 to soil applied zinc on site 4 for foliar zinc. The x-axis is pg Zn per shoot, the y-axis is pg Zn g-i and the dashed line is foliar mass per shoot in grams. The arrow indicates the direction of the response. Treatments are in kg Zn ha-1. 90 iii. Comparison of Foliar Versus Soil Treatments Foliar Zn treatments from all sites were more efficient in supplying the plant with Zn (Figure 17). This was calculated as the change in foliar Zn concentration (of treated minus control values) per gram of Zn applied per tree. In addition, the increase in foliar Zn occurred in the first year with the foliar Zn applications whereas, it was delayed until the second year with the soil Zn applications. The lower foliar Zn treatment (360 mg Zn L-1) was more efficient in supplying the plant with Zn than the higher foliar Zn treatments on all sites. b. Manganese i. Foliar Manganese Treatments The nutrient response to Mn tended to follow an opposite trend as compared with the Zn response. Response to foliar-applied Mn occurred in the second year only on sites 4 and 5, both to treatment 17 (4095 mg Mn L-1). On site 4 the increase in growth resulted in a manganese dilution (Figure 18A) (shift in A direction), and on site 5 the reduction in growth and Mn concentration is evidence of a manganese toxicity (concentration effect, shift in F direction) (Figure 18B). 91 Figure 17. Nutrient efficiency of foliar treatments in supplying the plant with Zn 2700 and 3600 are in mg 200 are in kg Zn ha-1-. Zn concentration (pg g Zn versus soil Zn Treatments 360, 1800, Zn L-1, and treatments 10, 50, 100 and Nutrient efficiency is change in foliar _1) per gram of Zn applied per tree. 92 Foliar Maas M 1800 n g a n e s e C o n 1700 1600 1500 1400 0.4 A ^4095 / — i I \ / <2>30 600 600 700 800 900 Manganese Content Figure 18. Vector diagrams of the second year growth response in 1987 to foliar applied Mn on site 4 (A) and on site 5 (B) for foliar Mn. The x-axis is pg Mn per shoot, the y-axis is pg Mn g_l and the dashed line is foliar mass per shoot in grams. The arrow indicates the direction of the response. Treatments are mg Mn L"1- . 93 ii. Soil Manganese Treatments In contrast, Mn nutrient concentration response to the soil Mn treatments occurred on all sites at all levels of treatments except for treatment 4 (200 kg Mn ha-1) on site 4 (Table 12). This response was still evident in the second growing season following manganese fertilization for all manganese treatments and sites. This was luxury consumption on sites 1, and 2 from treatment 3 (200 kg Mn ha-M in the first and second years, from treatment 3 (200 kg Mn ha"M on site 3 in the second year, on site 4 from treatment 5 (400 kg Mn ha_x) in the first and second years, from treatment 6 (600 kg Mn ha-1) in the first year, and on site 5 from treatments 4 (200 kg Mn ha"1), 5 (400 kg Mn ha-*) and 6 (600 kg Mn ha-1) in the second year (Table 12) . Growth responses to soil-applied manganese were obtained on sites 4 and 5. On site 4 a growth response was not detected until in the second year from treatment 6 (600 kg Mn ha-1) (shift in C direction) (Figure 19A). With treatment 4 (200 kg Mn ha-M, a Mn toxicity was obtained (shift in E direction), but at the highest Mn level (treatment 6 (600 kg Mn ha~M), a positive growth response to manganese was obtained (shift in C direction). Response on site 5 was different in that a positive growth response to manganese was evident in the first season. 94 Table 12. Foliar manganese (ug g-1) concentration and foliar mass per shoot (g) growth response to soil treatments of manganese. Values in parenthese are the standard deviation. An (*) indicates a significant difference and n.s.d. indicates no significant difference at the 5% level between a treatment and its respective sulfur control. Site 1 First Year Treatment Mn Hass -pg g_l- —g — 200 kg Mn ha"1 3008 0.360 (1298) (0.246) Control 1002 0.283 (306) (0.127) * n.s.d. Site 2 200 kg Mn ha"1 3142 0.145 (1470) (0.056) Control 1449 0.143 (655) (0.049) * n.s.d. Site 3 Treatment 200 kg Mn ha"1 Control Second Year Mn Mass -Pg g-1 g~ 2427 0.308 (441) (0.175) 1505 0.262 (287) (0.085) Second Year Mn Hass "Pg g"1" —g— 2971 0.348 (1278) (0.192) 1139 0.195 (395) (0.131) * n.s.d. 3875 0.181 (1429) (0.096) 1932 0.186 (589) (0.096) * n.s.d. Table 12 (concluded) Site 4 First Year Treatment Mn Mass "PS o~l- —S— 200 kg Mn ha"1 2026 0.270 (727) (0.096) 118 kg S ha~l 1639 0.271 (597) (0.178) n.s.d. n.s.d. 400 kg Mn ha-1 2802 0.352 (710) (0.388) 235 kg S ha"1 1647 0.275 (604) (0.058) * n.s.d. 600 kg Mn ha-1 3206 0.363 (1341) (0.284) 352 kg S ha"1 1175 0.287 (214) (0.239) Second Year Mn Mass — pg g"1 g — 2980 0.449 (918) (0.219) 1598 0.665 (412) (0.427) * * 3460 0.506 (780) (0.358) 1743 0.457 (471) (0.350) * n.s.d. 3737 0.646 (1237) (0.459) 1593 0.248 (365) (0.316) Site 5 First Year Treatment Hn Hass "PS g~l" ~g— 200 kg Mn ha"1 1832 0.302 (510) (0.096) 118 kg S ha"1 1083 0.193 (363) (0:075) * * 400 kg Mn ha"1 2172 0.250 (794) (0.134) 235 kg S ha"1 1268 0.168 (408) (0.037) * * 600 kg Hn ha"1 2933 0.367 (1021) (0.187) 352 kg S ha"1 1240 0.250 (526) (0.210) Second Year Mn Mass --pg s-t- ~g— 2487 0.317 (592) (0.089) 1359 0.291 (523) (0.156) * n.s.d. 2817 0.340 (651) (0.157) 1626 0.326 (632) (0.152) * n.s.d. 3518 0.316 (881) (0.103) 1301 0.314 (454) (0.180) * n.s.d. 96 (xWOO) Foliar Mass a t I o n 1' 1—— 1 1 1 500 1000 1500 2000 2500 Manganese Content Figure 19. Vector diagrams of the growth response to soil applied Mn for foliar Mn. (A) In the second year (1987) on site 4 and (B) in the first year (1986) on site 5. The x-axis is pg Mn per shoot in, the y-axis is pg Mn g-1 and the dashed line is foliar mass per shoot in grams. The arrow indicates the direction of the response. Treatments are in kg Mn ha"1. 97 A positive growth response to manganese occurred at all levels of treatment (shift in C direction) (Figure 19B). c. Complete-Zn-Hn Treatment i. Nutrient and Growth Responses A complete treatment was carried out in order to assess whether other nutrient deficiencies were present. The results are summarized in Table 13. On both sites 4 and 5, growth and nutrient concentration responses were produced in both the first and second years to the "complete -Zn -Mn" treatment. There was a positive growth response from the "complete-Zn-Mn" treatment on sites 4 and 5 in the first and second years (Table 13 and Figures 20 and 21) (shift in C direction). On site 4 in the first year (1986) the severity of nutrient deficiencies ranked from most to least deficient were in the order Zn, N, and B (Figure 20). On site 5 in the first year (1986) the severity of nutrient deficiencies ranked from most to least deficient were in the order B, Zn, AFe, Cu, Fe, and N (Figure 21A). In the second year the severity of nutrient deficiencies were in the order P, Zn and N (Figure 21B). Since there was a Zn response on sites 4 and 5 to the "complete-Zn-Mn" treatment, the Zn response was a synergism to the application of other nutrients (shift in C direction). 98 Table 13. Foliar nutrient concentrations and foliar mass per shoot growth response in unfertilized (control) trees and trees subjected to the "complete-Zn-Mn" treatment in the first and second years following treatment. An (*) indicates a significant difference at the 5% level of significance. s Ca eg £ Site 4 1986 Complete -Zn-Mn 1.67 0.16 0.67 0.30 0.12 CO.26) (0.014) (0.108) (0.017) (0.022) Control 1.0 0.20 0.68 0.28 0.14 (0.06) (0.071) (0.195) (0.088) (0.062) Hn Fe Active Fe Zn HE S~l  Cu 1343 38.4 37.2 (400) (1.8) (2.4) 1428 25.0 32.0 (360) 17.2 3.5 33.2 (6.2) (0.6) (4.1) 8.7 3.2 24.5 (1.9) (1.8) Site 4 1987 Complete -Zn-Hn 1.03 0.18 0.56 0.21 0.10 (0.15) (0.024) (0.094) (0.053) (0.029) Control 0.94 0.12 0.57 0.23 0.09 (0.21) (0.041)(0.112) (0.088) (0.040) Site 5 1986 Complete -Zn-Mn Control 1.67 0.21 0.81 0.30 (0.22) (0.066) (0.115) (0.088) 1.11 0.18 0.78 0.32 (0.11) (0.052) (0.042) (0.061) 0 .136 (0 .052 0.14 (0.016 Site 5 1987 Complete -Zn -Mn 1.32 0.24 0.86 0.34 0.15 (0.15) (0.037) (0.225) (0.085) (0.040) Control 1.15 0.17 0.84 0.31 0.13 (0.017) (0.062) (0.177) (0.072) (0.024) 1259 51.2 (305) (10.6) 1545 48.8 (415) (15.4) 1064 46.4 34.8 (370) (9.1) (6.4) 1308 39.3 26.2 (531) (6.7) (5.3) 962 53.7 (387) (7.0) 1243 52.7 (666) (12.0) 11.1 2.5 (3.5) (0.6) 11.7 2.9 (4.6) (0.6) 15.5 4.0 45.8 (4.2) (0.6) (10.9) 11.4 3.1 28.4 (1.9) (0.6) (6.0) 14.7 3.6 (2.3) (0.7) 11.8 3.9 (3.6) (0.9) Table 13 (continued). N/Pi H/P P/Al K/Ca Ca/Mg Site 4 1986 Complete -Zn -Mn Control Site 4 1987 Complete -Zn -Mn Control Site 5 1986 Complete -Zn -Mn Control Site 5 1987 Complete -Zn -Mn Control 10.4 10.3 4.3 2.2 2.6 5.1 6.2 4.5 2.5 2.0 5.9 6.4 7.6 4.6 7.3 3.1 7.8 10.3 6.1 6.9 5.5 8.2 6.9 7.1 4.9 3.3 4.4 2 . 7 2.2 2.3 2.7 2.4 2.6 2.7 2.2 2.1 2.2 2.3 2.3 2.4 Site 4 1986 Complete -Zn-Mn Control S (cg g-1) 0.173 (0.029) 0.160 H/S 9.65 6.25 Site 5 1986 Complete -Zn-Mn Control 0.114 (0.048) 0.124 (0.047) 14.65 8.95 1. Critical value calculated according to the formula. 100 Table 13 (concluded). Foliar Mass Per Shoot 1986 1987 g  Site 4 Complete -Zn -Mn 0.804 0.635 (0.646) (0.346) Control 0.200 0.280 (0.127) (0.145) Site 5 Complete -Zn -Mn 0.394 0.742 (0.162) (0.306) Control 0.198 0.288 (0.054) (0.140) * * 101 Site 4 1986 Figure 20. Vector diagram of the first year (in 1986) growth response to the "complete-Zn-Mn" treatment on site 4. The x-axis is relative nutrient content per shoot, the y-axis is relative nutrient concentration and the dashed line is relative foliar mass per shoot. The arrow indicates the direction of the ^ response. The control = 100. 102 Figure 21. Vector diagrams of the first (in 1986) (A) and second year (in 1987) (B) growth responses to the "complete-Zn-Mn" treatment on site 5. The x-axis is relative nutrient content per shoot, the y-axis is relative nutrient concentration and the dashed line is relative foliar mass per shoot. The arrow indicates the direction of the response. The control = 100. 103 The Increase in foliar Zn suggests that additional Zn was required with fertilization, and the site was able to meet these requirements. Referring to Table 13 in the first year on site 4, the control foliar Zn concentration was 8.7 ug g-1, and was increased to 17.2 ug g-1 in response to the "complete-Zn-Mn" treatment. Following the second year it was 11.7 ug g-1 and the treated level was 11.1 ug g-x• On site 5 in the first year the control foliar Zn concentration was 11.4 ug g~x and increased to 15.5 jig g~x with the "complete-Zn-Mn" treatment. In the second year the control level was 11.8 pg g-1 and the treatment increased foliar Zn to 14.7 pg g_x. It is interesting to see how the pattern of Zn response follows the pattern of nitrogen response. They both occurred only in the first year on site 4 and in years one and two on site 5. -There was not a great difference in foliar N levels for the second year controls on sites 4 and 5, 1.17 and 1.15 cg g~x respectively, yet there was a residual N response on site 5. 2. Shoot Increment Ratio For all the soil Zn applications on all the sites, a growth response occurred only on site 2 to treatment 2 (50 kg Zn ha-t) in the second year (Table 14). 104 Table 14. Shoot increment ratio growth response to zinc. Values in parenthese are the standard deviation. An (*) indicates a significant difference and n.s.d. indicates no significant difference at the 5% level between a treatment and its respective sulfur control. Site Tear Treatment Shoot Increment Ratio 2 2 SO kg Zn ha"1 1.58 (0.53) 25 kg S ha"1 1.14 (0.53) * 1 2 3600 mg Zn L"1 1.61 (0.71) 1770 mg S L-1 0.87 (0.23) * 4 2 360 mg Zn L~l 1.86 (1.22) 177 mg S L"1 1.14 (0 .39) 1800 mg Zn L"1 1.75 (0.70) 883 mg S L"1 1.46 (0.20) 2700 rag Zn L-1 1.43 (0.32) 1325 mg S L"1 1.30 (0.34) n.s.d. 5 2 360 mg Zn L_1 177 mg S L~l 1800 mg Zn L 883 mg S L-1 2700 mg Zn L"1 1325 mg S L"1 1.91 (0.89) 1.20 (0.29) 1.60 (0.54) 1.38 (0.41) n.s.d. 1.77 (0.43) 1.29 (0.54) 3 1 360 mg Zn L-1 177 mg S L-1 3600 mg Zn L~l 1770 mg S L-1 2 360 mg Zn L"1 177 mg S L-1 1.18 (0.20) 0.86 (0.27) * 0.78 (0.38) 0.82 (0.23) n.s.d. 1.02 (0.49) 0.86 (0.37) n.s.d. 3600 mg Zn L"1 1.02 (0.32) 1770 mg S L-1 0.79 (0.30) 105 Positive growth response to foliar Zn applications occurred only in the second year. On site 1, it took place at the highest application rate (treatment 5 (3600 mg Zn L_t)) (Table 14). On site 4 it was detected with treatments 13 (360 mg Zn L~l) and 14 (1800 mg Zn L~M (Table 14), and on site 5 with treatments 13 (360 mg Zn L-t) and 15 (2700 mg Zn L"M(Table 14). On site 3 it occurred in both years but to different levels of Zn (Table 14). In the first year, response took place to treatment 4 (360 mg Zn L_M, and in the second year to treatment 5 (3600 mg Zn L~M. Positive growth response to soil-applied Mn occurred on sites 4 and 5 in the first year (Table 15). On site 4 response occurred to all Mn treatments. On site 5, growth response was significant only to the 400 kg ha-1- treatment. Positve growth response occurred to the "complete-Zn-Mn" treatment on both sites 4 and 5 (Table 16) in the first year, and in the second year on site 5. 106 Table 15. Shoot increment ratio response to soil applications of manganese in the first year (1986) on sites 4 and 5. An (*) indicates a significant difference and n.s.d indicates no significant difference at the 5X level between a treatment and its respective sulfur control. Site 4 Site 5 Treatment Shoot Increment Ratio -200 kg Mn ha"1 1.03 (0.26) 1.25 (0.35) 118 kg S ha"1 0.83 (0.27) 1.09 (0.35) * n.s.d. 400 kg Mn ha"1 1.15 (0.38) 1.21 (0.52) 235 kg S ha"1 0.85 (0.28) 0.98 (0.37) * * 600 kg Mn ha"1 1.18 (0.37) 1.23 (0.39) 352 kg S ha"1 0.90 (0.32) 1.08 (0.39) * n.s.d. Table 16. Shoot increment ratio response to the "complete-Zn-Mn" treatment. An (*) indicates a significant difference and n.s.d indicates no significant difference at the 5% level between a treatment and its respective sulfur control. Site Year Treatment Shoot Increment Ratio 4 1 Complete-Zn-Mn 1.23 (0.48) Control 0.76 (0.22) * 5 1 Complete-Zn-Mn 1.58 (0.67) Control 0.96 (0.27) * 5 2 Complete-Zn-Mn 1.94 (1.11) Control 1.39 (0.14) 108 3. Height Increment Where growth response to Zn in terms of height growth increment occurred, it was not evident until in the second year (Table 17). On site 1 there was a significant response to treatment 5 (3600 mg Zn L"M, and a trend to response on site 2. On site S, the response occurred from treatment 13 (360 mg Zn L-M, and on site 4 there was a trend towards a response. In the first year of treatment for these sites the Zn treatments tended to depress growth. Height increment response to foliar Mn treatments did not occur until in the second year (Table 17). This was found on site 5 for treatment 17 (4095 mg Mn L~*). On site 5 response to soil Mn treatments occurred in the first year to treatments 4 (200 kg Mn ha-*) and 6 (600 kg Mn ha"1). On sites 4 and 5 in the second year there was a trend towards a response to treatments 4 (200 kg Mn ha-1) and 5 (600 kg Mn ha"1) respectively. C. Retranslocation Zinc and manganese concentrations were compared separately between first year foliage from 1985 and 1986, and between one-year-old and two-year-old foliage from sites 1 and 2 (Appendix M). For foliar Zn from the low treatment (treatment 4) there was a decrease in the two-year-old foliage but levels in the foliage formed in the second year were not different from the control 109 Table 17. Height increment ratio response. An (*) indicates a significant difference and n.s.d. indicates no significant difference at the 5% level between a treatment and its respective sulfur control . Site Tear Treatnent Height Increment Ratio I 2 360 mg Zn l"1 1.15 (0.61) 117 mg S L"1 0.97 (0.73) n.s.d. 3600 mg Zn L"1 2.54 (1.14) 1770 ng S L-l 0.83 (0.30) 2 2 360 mg Zn L-1 1.57 (0.85) 117 mg S L"1 1.42 (0.41) n.s.d. 3600 mg Zn L"1 1.85 (1.62) 1770 mg S L"l 1.57 (0.20) n.s.d. 4 2 360 »g Zn L"1 3 .30 (3 , .02) 117 mg S L"1 2 n . 35 .s.d , (1 , .66) 5 2 360 mg Zn L-1 2 .36 (1 . .28) 117 M S L-1 1 .43 * (0. ,46) 200 kg Mn ha"1 1 . .82 (0. , 76 ) 118 kg S ha"1 1 , .24 * (0 , . 46 ) 400 kg Mn ha"1 1 , .52 (0. .71) 235 kg S ha"1 1 . , 69 (0 . .87) n . .s.d. 600 kg Mn ha"1 1 . 74 (0. .53) 352 kg Mn ha"1 1 , .27 * (0. .57) 4095 mg Mn L"1 2 .22 (1 . .49) 2408 mg Mn L~1 I .26 (0 . .67) 110 (Table 18). The foliar Zn levels for the high treatment (treatment 5) decreased for the two-year-old foliage, and were elevated for the foliage formed in the second year relative to the control (Table 19). The increased Zn concentration in the foliage formed in the second year and the decrease in the two-year-old foliage suggests that there was retranslocation in the second year, of Zn from the old to the new foliage. This depended upon the level of the foliar Zn treatment, with remobilization occurring for the high foliar Zn treatment. On site 1 the Zn .retranslocation was associated with a growth response in terms of shoot and height increment ratio (Tables 14 and 17, respectively). Manganese levels were higher in the two-year-old foliage than the one-year-old foliage (Table Z0). The foliage formed in the second year also had elevated levels of Mn relative to the control (Table 20). Both these results indicate that the two-year-old foliage, as well as the current year's foliage formed in the second season following fertilization, continued to take up Mn. This suggests that increased uptake will occur if soil Mn is available. In the study of nutrient retranslocation it' was assummed that there was no change in unit foliar mass with foliage age. Therefore, foliar nutrient concentrations would no be changed from the dilution or concentrations effects. There is evidence which indicates that the decrease in foliar nutrient Ill Table 18. Change in foliar zinc (ug g~M of current year's foliage over time and with age of foliage following treatment for the low zinc foliar treatment on sites 1 and 2. Treatment 4 is 360 mg Zn L~x and treatment 7 is 177 mg S L~x. Site 1 Time Year since treatment Treatment Zn 4 Control 7 t-test Site 2 1 2 t-test 51.2 13.6 * 5.1 11.5 * * n.s.d Age Age since treatment 51.2 5.1 * 2 48.7 19.8 * t-test n.s.d * Time Year since treatment Treatment 1 2 t-test Zn 4 53.0 11.5 * Control 7 2.0 8.8 n.s.d t-test * n.s.d Age Age since treatment 53.0 2.0 * 2 35 .2 15.6 * t-test * n.s.d 112 Table 19. Change in foliar zinc (pg g~M of current year's foliage with time and with age of foliage following treatment for the high zinc foliar treatment on sites 1 and 2. Treatment 5 is 3600 mg Zn L-1 and treatment 8 is 1770 mg S L-t. Site 1 Time Age Years since treatment Age since treatment Treatment 1 2 t-test 1 2 t-test Zn 5 144.4 15.4 * 144.4 135.5 * Control 8 2.5 11.0 * 2.5 17.9 * t-test * * * * Site 2 Time Age Years since treatment Age since treatment Treatment 1 2 t-test 1 2 t-test Zn 5 139.6 19.6 * 139.6 78.7 * Control 8 2.5 11.4 * 2.5 17.0 * t-test * * * * 113 Table 20. Change in Collar manganese (pg g~M of current year's foliage with time and with age of foliage following treatment for the manganese soil treatment (3) on sites 1 and 2. Treatment 3 is 200 kg Mn ha-1 and treatment 12 is the untreated control. Site 1 Time Years since treatment Treatment 1 2 t-test Mn 3 3059 2971 n.s.d Control 12 1019 1139 n.s.d t-test * * Age Age since treatment 1 3059 1019 * 2 4610 1432 * t-test * Site 2 Time Years since treatment Treatment 1 2 t-test 1 Mn 3 3196 3875 * 3196 Control 12 1474 1932 * 1474 Age Age since treatment 2 5920 2365 t-test * * t-test I 114 concentrations in the two-year-old foliage was not due to foliar leaching. With an increase in foliage age, foliar Zn concentrations for the controls increased (Tables 18 and 19) indicating no evidence of Zn leaching. D. Nutrient-Growth Interactions Data were fitted to the model y = a + bx + cx2, where y is a measure of growth and x is foliar Zn concentration, to test the hypothesis that the relationship of foliar Zn concentration to growth follows the model of diminishing returns. Significant regressions were found between height increment and foliar Zn concentrations (Figure 22 and Table 21). These were found between current year's height increment and foliar Zn for 1986 and 1987 for sites 4 and 5. Comparing the foliar levels of the optimum height increment to the control Zn levels suggests that height increment benefited from the higher foliar Zn concentrations. There were no relationships found between other growth parameters either measured or calculated and foliar nutrient concentrations. 115 Figure 22. Scatter plots of height increment (cm) versus current year's foliar zinc levels (pg g-1). (A) In the first year (1986) (for cases where Zn <50 pg g-1) and (B) in the second year (1987) for site 4. 116 Table 21. Equations and correlation coefficients for the height increment (y) - foliar zinc (x) relationships for sites 4 and 5. Refer to Appendix N for scatter plots other than given in Figure 22. Site Year Equation Ra 4 1986 y • 5.125x - 0.094xa 0.81 4 1987 y = -31.677 + 15.209 - 0.481xa 0.37 5 1986 y = 2.709x - 0.03xa 0.22 5 1987 y = 8.577x - 0.234xa 0.18 117 E. Nutrient Interactions 1. Interactions with Zinc a. Manganese On site 5 in the first year, treatment 15 (2700 mg Zn L~M produced the only incident where Zn application reduced foliar Mn (Table 22). Treatment 1 (50 kg Mn ha-1) actually increased foliar Mn. b. Nitrogen Scatter plots of foliar N versus foliar Zn revealed significant relationships between the two (Figure 23 and Table 23). In addition, a linear relationship was found to exist between extractable soil Zn and total soil N concentrations (Figure 24). 118 Table 22. Foliar manganese (pg g"*) nutrient concentration response to soil and foliar applications of zinc in the first, year (1986) on site 5. Soil Treatments Foliar Treatments Treatment Mn Treatment Mn 50 kg Zn ha"1 1262 Q37) 25 kg S ha~* 1178 (416) 100 kg Zn ha"* 1273 (209) 49 kg S ha"* 1525 (670) n.s.d. 200 kg Zn ha-* 1289 (548) 98 kg S ha~* 1327 (536) n.s.d. 360 mg Zn L~* 1217 (235) 117 mg S L-* 1140 (249) n.s.d. 1800 mg Zn L~* 1066 (319) 883 mg S L~* 1272 (751) n.s.d. 2700 mg Zn L~* 867 (487) 1325 mg S L~* 1094 (261) * 119 1.60 -i OO 1.20 -CT> C CU ?0.80 H 0.40 -y = 0.52 + 0.076x - 0.00162x: Ra = 0.52 (B) 0.00 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 0.0 10.0 20.0 30.0 Foliar Zinc 1986 11111 40.0 Figure 23. Scatter plots of foliar nitrogen (cg g-1) versus foliar zinc (pg g-1) of the current year's foliage (A) in the first year (1985) (for cases where Zn <50 pg g-1) and (B) in 1986 (for cases where Zn <40 pg g-1) from site 1. The correlation coefficient (R2) is given. 120 Table 23. Equations and correlation coefficients (Ra) for foliar nitrogen (y) - foliar zinc (x) relationships according to site. Refer to Appendix 0 for scatter plots other than given in Figure 23. Site Year Equation R2 1 1985 y • 0.52 + 0.07x - 0.00162xa 0.52 1 1986 y • 0.6 + 0.047x - 0.0009xa 0.21 2 1985 y = 0.6 + 0.0997x - 0.0033txa 0.44 2 1986 y = 0.8 + 0.038x - 0.0009xa 0.21 4 1986 y = 0.l37x - 0.0036lxa 0.46 4 1987 y - 0.55 + 0.074x - 0.00134xa 0.50 5 1986 y = 0.306 + 0.0932x - 0.00l76xa 0.65 5 1987 y • 0.234 + 0.115x - 0.00293xa 0.54 121 Figure 24. Scatter plot of total soil nitrogen (eg g-1) vesus extractable soil zinc (ug g-1-) both from the forest floor for all sites . 122 2. Interactions with Manganese a. Zinc Manganese foliar and soil treatments affected foliar Zn levels on site 1 (Table 24). Foliar-applied Mn increased foliar Zn in both the first and second years. The Mn soil treatment (200 kg Mn ha~M increased foliar Zn in the first year. On site 5, foliar Zn was also increased by soil applications of Mn in the first year from treatments 4 (200 kg Mn ha~M and 6 (600 kg Mn ha-1) (Table 25). As shown in Figure 25 this was a synergism of foliar Zn to Mn applications (shift in C direction). There were no other relationships found between Mn treatments and foliar concentrations of other nutrients. Therefore, it is unlikely that the growth responses obtained with Mn soil treatments were due to the alleviation of toxicities of other nutrients or metallic elements. Table 24. Foliar zinc (ug g"M nutrient concentration response to soil and foliar manganese applications on site 1. Foliar Treatment Treatment 1985 1986 Zn Zn ug g-l. 2730 mg Mn L"v 9.4 (2.5) 10.6 (1.5) Control 7.2 (1.7) 8.8 (2.3) * * Soil Treatment Treatment 1985 Zn -ug g~x-200 kg Mn ha"1 15.1 (5.8) Control 7.6 (1.9) Table 25. Foliar zinc (ug g-1) nutrient response in the first year (1986) to soil applications of manganese on site 5. Soil Treatments Treatment 200 kg Mn ha"1 118 kg S ha"1 400 kg Mn ha'1 235 kg S ha"1 600 kg Mn ha-1 352 kg S ha"1 Zn -ug g-1— 11.9 (2.3) 8.3 (1.5) * 12.1 (5.2) 10.5 (2.7) n.s.d. 15.7 (5.8) 10.1 (1.4) 125 Figure 25. Vector diagram of first year growth response (in 1986) of Zn to soil treatments of Mn from site 5. The x-axis is ug Zn per shoot, the y-axis is ug Zn g-1 and the dashed line is foliar mass per shoot in grams. The arrow indicates the direction of the response. Treatments are in kg Mn ha-1. 126 F. Relationship of Response to Site A summary of nutrient and growth responses to foliar and soil Zn treatments are presented in Tables 26 and 27 respectively. There was no obvious relationship between response and site conditions. In similar manner, for the soil Mn treatments (Table 28) there was no relationship between response and site. An interesting relationship was found between site conditions and response to the "complete-Zn-Mn" treatment- on sites 4 and 5 (Table 29). There are distinct differences between sites 4 and 5. Site 4 is CWHbl, low elevation and has a humo-ferric podzol with an Ae horizon. In contrast site 5 is CWHb2, high elevation and has a ferro-humic podzol with an enriched organic B horizon. There was a response to N, Zn and B in the first year regardless of site (Figures 20 and 21A). On site 5 response to N and the Zn synergism continued into the second year (Figure 21B). In addition there were responses to Cu and Fe in the first year (Figure 21A) and a strong response to P in the second year (Figure 21B). 127 Table 26. Summary of nutrient and growth responses to foliar Zn treatments. A (*) indicates a significant increase, a (**) indicates a significant decrease and a (-) indicates no significant difference at the 52 level between a treatment and its control. First Year Second Year Site Treatment Zn Mass Shoot Height Zn Mass Shoot Heigh (mg Zn L-l) Ratio Ratio Ratio Ratio 1 360 * 3600 * — — — * — * * 2 360 * * — - - — - -3600 * ** - — * — — — 3 360 * - * * - -3600 — — — — * — 4 360 * - - - - - * -1800 * - - - - - * -2700 * — - - - — — — 5 360 * ** — — _ — * — 1800 * - - - - - - * 2700 * - - - - - * -128 Table 27. Summary of nutrient and growth responses to soil Zn treatments. An (*) indicates a significant increase and a (-) indicates no significant difference at the 5X level between a treatment and its control. Second Year Site Treatment Zn Hass Shoot Height (kg Zn ha-1) Ratio Ratio 10 * 5010 50 10 50 * 50 100 200 * 50 100 200 * 129 Table 28. Summary of nutrient and growth responses to soil Zn treatments. An (*) indicates a significant increase, a (**) indicates a significant decrease and a (-) indicates no significant difference at the 5% level between a treatment and its control. First Year Second Year Site Treatment Mn Mass Shoot Height Mn Mass Shoot Height (kg Mn ha-l) Ratio Ratio Ratio Ratio 1 200 * - - - * - - • 2 200 * - - - * - _ 3 200 - - * - -4 200 — — * — * ** — — 400 * - * - * - -600 * — * - * * — — 5 200 * * — * * _ - — 400 * * * - * - -600 * * - * * - -130 Table 29. Summary of nutrient and growth responses to the "complete-Zn-Mn" treatment. An (*) indicates a significant increase and a (-) indicates no significant difference at the 5% level between a treatment and its control. First Year Site N P Zn Fe Active Fe Cu B Mass Shoot Height Ratio Ratio 4 *_*_ _ - ** * 5 * — ** * *** * P Second Year Zn Mass Shoot Ratio Height Ratio 4 * 5 * * 131 CHAPTER 5. DISCUSSION A. COMPARATIVE NUTRITION 1. Total Levels Hemlock had lower foliar Zn concentrations and higher foliar Mn concentrations compared to Douglas-fir, amabilis fir and red cedar. Zasoski er al. (1990) also reported foliar Zn concentrations to be lower and foliar Mn concentrations to be higher than some other conifers in the Pacific Northwest. The fact that the comparison in this study was made among species on the same sites and in the same stands would indicate that the levels of Mn and Zn in hemlock are plant-related and not soil-related. In addition, it is unlikely different levels of foliar Zn and Mn between species are due to the exploitation of different soil horizons by the root systems. Root excavations done by Eis (1987) found that on similar soils, roots of Douglas-fir, cedar and hemlock trees penetrated to similar depth and trees of similar size extended their root system over similar areas. Since there were no significant differences in the rooting depth between species of 50-year-old trees, it may be safe to assume that the rooting depth of species of younger trees, as used in this study, would not be significantly different. Even if hemlock was exploiting the forest floor more than other species, this part of the soil horizon has the highest available Zn (Table 30). Table 30. Soil data for all sites. 'T* refers to the top horizon which was forest floor and 'B' refers to the top 15 cm of the mineral soil horizon. MEAN SITE ZnT ZnB Mnl MnB KT KB AIT Al B MS g-1  1 22.1 2.9 49 .6 5 . 1 196 .1 55 . 4 1116 1707 3 15.2 1.8 226.0 13. 6 431 .0 188 .6 1037 1758 4 29.2 5.8 44.4 21. 0 198 .4 166 .0 874 1495 5 23 . 7 1.8 47.6 2 . 8 249 .6 28 .6 1176 1762 STANDARD DEVIATION 1 17.6 1.6 40 .2 6 . 5 121 .4 41 .8 438 289 3 6.7 1.3 51.3 3. 1 190 .5 45 .2 364 343 4 10.6 3.6 21.8 35 . 9 48 .1 95 .5 325 210 5 13.4 0.7 27.6 2 . 3 204 .0 14 .5 425 541 MEAN SITE CaT CaB MgT MgB PT FB CuT CuB b 1 1 879 80 208 23 . ,2 56 . 3 12 . 7 0.96 0.40 3 1175 210 350 48 . , 7 58. 0 6. 9 0 .64 0.56 4 1447 233 267 64. .0 70 . 3 17 . 5 1 .32 0 .82 5 1268 137 288 22 . .1 72 . 6 7 . 2 1.22 0 .96 STANDARD DEVIATION 1 688 77 178 21. .2 53 . 3 16 . 1 0.59 0 .26 3 416 113 17 20 . .6 26 . 7 5 . 8 0 .17 0 .16 4 541 200 145 56 . .2 25 . 4 18 . 1 0 . 22 0.50 5 706 68 141 6 . .9 48 . 8 7 . 8 0.50 0 .63 BT BB EXNT EXTNB TNT TNB pHT pHB g~l eg g-1 Ha0 0.47 0. 16 0 .016 0 . .004 0 . 60 0 .19 3 . 8 4 , . 1 0.40 0. 11 0 .063 0 .016 0 .67 0 .24 4 .5 4, .6 0.03 0 . 19 0 .013 0 .005 0 .90 0 . .24 4 .0 3 . . 8 0 . 14 0 . 05 0 .017 0 .005 0 .93 0 .20 4 . 1 4 , .5 0 . 78 0 . 06 0 , .021 0 . .002 0 , .38 0 . .10 0 , .3 0 . ,3 0.03 0 . 04 0 . .044 0. .010 0 , .07 0 , .11 0 . .3 0 . . 3 0.01 0 . 11 0 , .012 0 , .001 0 . .26 0 . .14 0 . .6 0 . , 7 0.09 0 . 06 0 , .020 .0 , .003 0 . .24 0 , .11 0 .3 0 . . 4 FeT FeB pHT pHB CECT CECB --Cf iCla' — -meq lOOg" 184 226 3 .5 3 .8 12 .9 14.2 261 285 3 .9 4 .1 6 . 7 19 .6 123 217 3 .4 3 . 7 7 . 7 13.2 179 177 3 .5 3 . 7 9 . 4 12.4 43 118 0 .4 0 . 4 6 . 1 2.8 37 252 0 . 1 0 .4 1 .2 7 . 4 38 114 0 . 4 0 .2 2 .8 . 6.9 89 93 0 . 3 0 .5 3 . 1 6 . 4 U) 133 The differences among species may be attributed to differences in the regulation of nutrient uptake and/or differences in the regulation of nutrient transport between root and shoot. Among the possible reasons for unusual nutrient concentrations of Zn and Mn may be different physiological requirements and/or ecological strategies in hemlock. A situation similar to hemlock has been found for black spruce and jack pine. Black spruce has higher foliar concentrations of manganese as compared with jack pine (Morrison and Armson 1968; Lafond and Laflamme 1968). This is an example of how the physiology of the plant is tied into its ecology. Morrison and Armson (1968) contend that the high Mn levels of the spruce foliage create Mn-rich surface layers which allow regeneration of spruce but inhibit the regeneration of jack pine. 2. Extractable Zn and Mn Douglas-fir has higher water-soluble Zn than hemlock but amabilis fir has similar levels of water-soluble Zn to hemlock. This may suggest that Douglas-fir has a higher component of active zinc, which might indicate a higher requirement for Zn than in hemlock and amabilis fir. The higher total and water-soluble Zn in white pine than hemlock which in turn had a higher level than cedar suggests a relative physiological requirement in the order pine>hemlock>cedar. 134 The higher level of water-soluble Mn in hemlock than Douglas-fir, amabilis fir, pine and cedar may indicate a higher requirement for Mn by hemlock. 3. Cellular Fractions of Foliage Fractions of foliage represent different cellular components which are involved in specific physiological processes. For example, fraction B and C represent the chloroplastic component which are involved in the process of photosynthesis. Hemlock had lower Zn in most fractions compared to Douglas-fir. This relationship is consistent with total foliar levels. Therefore total foliar Zn concentrations may reflect physiological levels of Zn. The method used to separate fraction E is similar to the method used by Rahimi and Schropp (1984), in which they measured the physiologically active Zn associated with carbonic anhydrase activity. Therefore the Zn level in the E fraction may be the physiologically active Zn. Hemlock had higher Mn levels compared to other species in all fractions. These results for Mn may have several interpretations. Higher levels may mean that hemlock has a greater physiological requirement for Mn, or alternatively may represent some Mn accumulation as a tolerance mechanism. I Therefore, indication total foliar Mn levels may not of physiologically active Mn. necessarily be an 135 The only other study which has examined Mn in cellular fractions of foliage was by Memon (1984). He examined the manganese concentration of different cell fractions in the foliage of a Mn accumulator. Manganese was highest in the supernatant fraction which in this study was part of fraction E. B. Fertilization Experiments 1. Nutrient and Growth Responses There were positive linear and curvilinear relationships between current height increment and foliar mass per shoot. There is evidence which supports a relationship between foliar mass and growth. For example, there is a curvilinear relationship between leaf area index and the net primary productivity. Net primary productivity reaches a maximum with a specific leaf area index but above this point yield is reduced because of high respiratory losses required to maintain a large volume of leaf and supporting tissues (Kramer and Kozlowski 1979). Ford (1984) reviews a study in which a positive correlation was found between annual branch-and-bole production 136 and foliage biomass for both conifers and deciduous trees. However, the study did not show as the authors claim that there is "no decrease in dry matter production even at the highest leaf biomass". To do this they should have demonstrated no evidence of a curvilinear relationship (Ford 1984). Therefore, it has not been proven that tree growth may continue to increase indefinitely as foliage amount increases (Ford 1984). Some equations fitted to data from this thesis suggest that there may be a decrease; however, unambiguous evidence has not been obtained. a. Zinc i. Comparison of Foliar Versus Soil Treatments The foliar and soil methods of application were used in order to test whether some factor(s) [e.g. of the soil, the uptake mechanism, or translocation] was interfering with the movement of soil-applied zinc to the foliage. The foliar hemlock nutrient data indicate the greater efficiency of Zn uptake with foliar applications compared to soil applications. Foliar applications of Zn have been found to be up to 12 times more efficient than soil applications (Murphy and Walsh 1972). Stout er al. (1987), found that with alfalfa, Zn recovery in the plant was less than 2% of the soil-applied Zn. Since in hemlock there was a delay in zinc response to soil applications, and the changes in foliar concentration were not as dramatic as the 137 foliar treatments, one or more of the above mentioned factors may have accounted for the pattern of response observed for soil Zn applications. In agriculture, zinc is applied to the soil for row crops and as a foliar spray for tree and vine crops (Traynor 1980). Chevis (1983) found that a 2.5 mg L-x zinc sulphate foliar spray raised the foliar zinc concentrations in Pinus radiata, correcting a deficiency, while soil applications of zinc oxide did not correct the deficiency. Seedlings of P. elliottii grown in pots took up zinc in the foliage when zinc was applied to the soil in a solution form (van Lear and Smith 1972). McKee (1976) found that seedlings of P. elliottii responded to zinc applied in solution form to the soil in a pot experiment. Seedlings of P. radiata did not display symptoms of zinc deficiency when it was supplied in solution culture (Smith and Bayliss 1942). McGrath (1978) concluded that application of zinc as a foliar spray was a more effective and reliable method of supplying zinc to P. radiata than addition to the soil of zinc oxide with superphosphate. Symptoms of zinc defir.i«ncy w«re ov«rr;ome when a IX solution of zinc chloride was applied to the foliage (Kessel and Stoate 1936). Foliar applications of 0.6 mg L~x zinc to seedlings of P. radiata in the nursery raised their foliar zinc concentrations and prevented visual symptoms of zinc deficiency (Knight 1975). A foliar application of 9,800 ug g-i of zinc chelate corrected the growth disorders of 46-month-old trees of P. caribaea (Sance et al. 1982). Stoate (1950) found that foliar 138 applications of 2.5 mg L-t zinc sulphate as a foliar spray or soil dressings of 0.12 grams to 1.8 kg zinc sulphate to individual trees prevented the continuation of growth disorders in P. radiata. Firstly, availability may have been affected by factors of the soil. Availability of Zn from the soil may be a problem due to transport from the soil to the root. Autoradiographs of wheat roots using ssZn have indicated zones of depletion around the roots. This depletion indicates the creation of a concentration gradient, and that the ion moves in part by diffusion (Wilkinson er al. 1968). Since diffusion is important in the transport of the ion, root contact with the soil is important in the absorption of zinc (Boawn et al. 1957). This is demonstrated by the fact that availability of zinc is increased when it is mixed with the soil rather than applied in a band (Shaw er al. 1954; Murphy and Walsh 1972). Wider distribution would increase its contact with roots (Murphy and Walsh 1972). The soil pH affects the availability of zinc through its solubility. Studies examining the influence of pH on zinc adsorption show a decrease in zinc solubility with increasing pH (Saed and Fox 1977; Bar-Yosef 1979; McBride and Blasiak 1979). The solubility of zinc decreases 100-fold for each unit increase in pH (Tisdale er al. 1985). With soil acidification, plant uptake of soil Zn or banded applications were increased (Viets er al. 1957). 139 In a study by Maclean (1974), corn, lettuce and alfalfa plants were grown with Zn levels similar to those found in the soils used in this study (Table 30). It is interesting to note that the soils had a DTPA-extractable Zn of 26.8 pg g-1, and a pH of 4.9 while the corresponding foliar levels of the plants were 238 (corn), 523 (lettuce), and 321 (alfalfa) pg g-1, which were toxic to growth. Since the soil Zn levels found by Maclean (1974) were toxic to plants, Zn availability in hemlock may be limited by uptake or some interaction but not by soil supply. Two additional sources of information suggest this. First, some other conifer species on the same sites have higher foliar Zn concentrations than hemlock. Second, increased uptake of Zn occurred with the "complete-Zn-Mn" treatment indicating additional soil Zn was available. Therefore, with the soil pH levels of the study sites being relatively low (3-4), the high inherent supply of available Zn, the use of broadcast applications, and the use of the soluble ZnSO*, it is unlikely that there was a problem of Zn fixation, insolubility or low soil Zn levels. The second possible reason involves the uptake and translocation of zinc. This involves two aspects; firstly the physiological requirements of the plant for Zn, and secondly the ecological aspect of metal tolerance. Hemlock may accumulate Zn in the roots and restrict transport from the roots to the shoots, 140 or restrict uptake from the soil to the root. Work done with several species [soybean (White er al. 1979), bush beans (Ruano et al. 1988; Hawf and Smith 1967), maize and barley (Singh and Steenberg 1974), maize (Nair and Prabhat 1977), various species (Carroll and Loneragan 1968), and subterranean clover (Riceman and Jones 1958)] indicates that with Zn application, Zn accumulates in the roots relative to the shoot. The delay in foliar response to soil applications is similar to the situation found by West (1979) with Cu in P. radiata. Roots tended to accumulate Cu, but over time there was a net decrease in root Cu levels and an increase in shoot Cu levels. It appears that some of the excess Cu, stored in roots which had been exposed to high Cu levels, was released to shoots when sufficient Cu was no longer available (West 1979). The results of the foliar and soil Zn treatments suggest that it is factors of the plant related to uptake and/or translocation which account for the observed pattern of response to Zn, and hence the characteristic Zn nutrition of hemlock. ii. Zinc Tolerance Of the three types of plant-soil nutritional relationships (accumulator, excluder and indicator), hemlock may be an excluder with respect to Zn, judging from the pattern of response to soil Zn treatments. Observations of metal-resistant plants 141 from different ecological situations show that tolerance is not achieved by exclusion with respect to uptake (Ernst,1975). Restriction of transport from root to shoot is the likely mechanism of control (Baker 1981; Woolhouse 1983; Foy er al. 1978). Consequently, metal concentrations in the shoot are maintained constant and low over a wide range of soil concentrations, up to a critical soil value above which the mechanism breaks down and unrestricted transport results (Baker 1981). This phenomenon of Zn exclusion has been examined in several species. For example, different populations of Silene maritima from Zn-contaminated soils, when grown in solution culture, excluded Zn from their shoots (Thurman 1981). Agrostis tenuis is a species of grass which has zinc-tolerant clones in which tolerance is conferred on the plant through increased retention of Zn in the roots (Woolhouse 1983). Zinc-tolerant clones of the grasses Deschampsia caespitosa and Anthoxanthum odoratum concentrated Zn in their roots (Brookes er al. 1981). Seedlings of hemlock grown in solution culture were found to have higher zinc concentrations in the roots compared to the foliage (Table 31) (Zasoski er al. 1990). In excluders, detoxification occurs largely within the roots (Baker 1981). The mechanisms by which Zn may be detoxified in the root are immobilization of Zn in cell walls and compartmentation as a soluble complex, free ion or insoluble complex (metallothionein) (Woolhouse 1983). 142 Table 31. Effect of solution pH on micronutrlent concentrations (mg kg-1) in Douglas-fir and western hemlock roots (R) and needles (L) (from Zasoski er al. 1990)). Solution pH 3 3 .5 4 4.5 5 Douglas -fir Fe R 424 666 1180 1810 2240 L 78 80 90 95 88 Mn R 21 27 25 27 30 L 87 97 100 102 97 Zn R 90 87 78 80 74 L 47 45 46 48 43 Cu R 53 84 95 129 144 L 28 32 30 34 31 Western Hemlock Fe R 301 779 1950 3290 3780 L 104 112 116 141 142 Mn R 34 37 37 39 42 L 265 2 70 266 289 256 Zn R 78 69 74 84 77 L 35 41 33 39 34 Cu R 54 63 78 110 117 L 54 55 60 66 63 143 Denny and Wilkins (1987a), studying zinc tolerance in Betula, rejected the tolerance mechanisms of internal detoxification and cell wall binding.' They found using microanalysis that at Zn concentrations above the lethal level, Zn accumulated intracellularly, at the endodermis, in the form of electron-dense granules. They also investigated the mechanism of ectomycorrhizal amelioration of zinc toxicity to Betula. As the fungal hyphae penetrate the soil, Zn is adsorbed to the surface of hyphae, thereby lowering the concentration of zinc in the soil solution surrounding the roots. The metal may also be adsorbed to electro-negative sites in the hyphal cell walls and to extra-hyphal, polysaccharide slime (Denny and Wilkins 1987b). Another method of detoxification has been investigated by van Steveninck er al. (1987). They found high levels of Zn in globules in the roots of zinc-tolerant ecotype of Deschampsia caespitosa. The globules appear to occur in small vacuoles within the cytoplasmic matrix of elongating cells in the cortex. An additional mechanism is that Zn may be concentrated by the mycorrhizae. Zinc has been found to be concentrated in the roots of mycorrhlzal plants of Pinus virginiana (Miller and Rudolph 1986), Betula (Brown and Wilkins 1985), and Ericaceous plants (Bradley er al. 1982) compared to uninfected plants. Since hemlock roots are known to be highly mycorrhlzal (Gill and Lavender 1983), it is possible that such a mechanism is operating in hemlock. b. Manganese 144 i. Comparison oE Foliar Versus Soil Treatments Nutrient responses to soil applications of Mn were much more efficient than foliar applications. In addition, responses were consistent with soil applications. The reasons for the different responses between Zn and Mn when applied as a foliar spray require an examination of the process of solute movement through the foliage as outlined in Figure 26. Foliar uptake of nutrients involves deposition, absorption through the cuticle, epidermal cells (Bukovac and Wittwar 1957), and the stomates (Swietlik and Faust 1984), adsorption on the surface of the plasma membranes, passage through the plasma membranes and movement into the cytoplasm (Swietlik and Faust 1984). Initial penetration of the cuticle occurs by diffusion, which depends upon temperature and a concentration gradient (Swietlik and Faust 1984). Manganese deficiency was corrected in P. radiata using foliar sprays with Mn at 8.5 and 8.75 mg L"1 compared to the foliar levels of 10 pg g~x (Ruiter 1983). The concentration of manganese in the solution may have been too small relative to the concentration in the foliage for diffusion to occur. In agriculture, foliar application of Mn is one of the most efficient ways of correcting Mn deficiency (Murphy and Walsh 1972), and MnS0«, which was used in this study, is considered to be the most effective inorganic carrier of Mn (Murphy and Walsh 1972). 145 Surface wetting , Cutfcular penetration Active transport across the plasma lemma ApopJastic free apace movement Synthesis of organic compounds Sympiastic movement via the piasmadesmata Energy-dependent phloem loading Energy-dependent phloem loading Translocation Figure 26. Possible pathways of solute movement through the leaf (from Haynes (1986)). 146 Therefore the lack of response to foliar applied Hn found in this thesis is probably not due to the application method per se or the type of carrier. Differences in the foliar uptake and translocation of Zn and Mn have been demonstrated in several studies. Bukovac and Wittwar (1957) found that 6BZn moved through the transpiration stream, whereas Mn tended to concentrate where it was applied and a greater percentage of the Zn was absorbed. Romey and Toth (1954) found B*Mn to be absorbed through the foliage although there were differences between plant species in the amount absorbed. But, Hn was again found to concentrate in the interveinal tissues, forming small islands. Chamel (1985) found Mn to be poorly absorbed, since discs which were initially charged with s*Mn lost almost all the applied Mn by washing, whereas 80% of the esZn was lost. He concluded that cuticular retention is dependent upon the element considered. Manganese has been found to be readily leached from foliage (Tukey er al. 1958). However, in another study with pea plants there was 100X absorption of Mn into the treated area of the leaf (Ferrandon and Chamel 1988). In this study, the characteristics of the element, the concentration of the solution, and the characteristics of the foliage may have all contributed to the low Mn absorption by the hemlock foliage. ii. Toxicity Levels 147 The results Indicate a growth response to soil-applied Mn. This is interesting because the foliar levels for the control trees are higher than toxicity levels reported for agronomic crop plants. Reported toxicity levels are 1,000 ug g_1 in beans, 550 pg g_1 in peas, and 200 pg g-i in barley (White 1970), 380 pg g~x in Medicago sativa, 1,600 pg g~x in Centrosena pubescens, > 2,600 pg g-x in carrots, > 160 pg g-x in Bragg soybeans, 450-500 pg g-x in tomato (young leaves) (Foy er al. 1978), 120-600 pg g-x in apple, 445-1400 pg g-t, respectively, in upper and lower leaves of sweet sorghum, 500 pg g-x in flax, 500-1,960 pg g-x in cotton, 2,000 pg g~x in Easter lily, 2,600 pg g-x in carnation, 2,500-6,500 pg g-x in maize (Foy 1983), 1,200-2,600 pg g-x in coffee, and 120 pg g"1- in snap beans (Foy 1984). There are several examples of Mn foliar levels and the occurrence of toxicity symptoms in forest trees. Dying European fir seedlings having blackened roots suggestive of a Mn toxicity had foliar levels of 1,300 to 2,500 pg g-x compared to 120 to 500 for living seedlings (Stone 1967). A 60% reduction in growth of black spruce and jack pine seedlings grown in solution culture was noted when the culture solution contained 100 pg g-x Mn, and the corresponding foliar Mn levels of the seedlings were 4,200 to 4,400 pg g-x (Morrison and Armson 1966). Although hemlock had higher or similar foliar Mn levels for the Mn soil treatments compared to the toxicity levels reported in the literature for other plants, there was no evidence of a Mn toxicity in this study. 148 iii. Manganese Requirements The foliar Mn levels are higher for hemlock than for other tree species in the same stands. In addition, there was a foliar nutrient concentration response to soil applications of Mn, without a reduction in growth. These facts suggest that hemlock is manganese-tolerant and has a greater total Mn requirement compared to some other B.C. conifers. This is consistent with other studies which have reported an increased need for metals in metal-tolerant ecotypes of many species. Examples of these are wheat (Mn) (Macfie 1989; Foy er al. 1973), copper moss (Scopelopbila cataractae) (Shaw 1987), Agrostis tenuis (Cu, Pb, Zn), Mimulus guttatus (Cu), Anthoxanthum odoratum (Zn), ffolcus lanatus (Zn), Armeria maritima (Zn), Silene vulgaris (Zn) (Antonovics 1971), Succisa pratensis (Mn) (Pegtel 1986), Avenella Flexuosa, Chamaenerion angustifolium, Rumex acetosella, and Senecio sylvaticus (Mn) (Ernst and Nelissen 1979). This phenomenon has been explained by invoking zinc-tolerant plants. If a tolerant plant complexes the metal in order to detoxify it, one would expect a shortage in these plants on normal soils (Ernst 1975). The biomass production would be diminished and can be stimulated by Zn concentrations which are already toxic for non-tolerant plants. The activity of carbonic anhydrase was found to be stimulated at high amounts of zinc in tolerant populations, but not in non-tolerant populations (Ernst 1975). Therefore, because of the efficiency of the tolerance mechanism 149 in inactivating the metals, the external trace element requirement is higher (Antonovics er al. 1971). vi . Manganese Tolerance Hemlock is similar to other plants growing on acidic substrates in that it is a manganese accumulator which enables it to tolerate this type of environment. These types of plants are classified as calcifuges, which are plants adapted to low soil pH. Ericaceae is a family of plants which like hemlock are calcifuges. Species in the Ericaceae belonging to the genus Vaccinium also occur in stands of hemlock. This is of interest because some species of Vaccinium are of agronomic importance and work has been done on their nutrition. Of the three types of plant-soil relationships, hemlock may be classified as an accumulator with respect to Mn. This is similar to the Vaccinium where internal tolerance especially in leaf tissue is the mechanism of tolerance (Korcak 1988). Foliar levels of Mn in Vaccinium species have been reported in excess of 2,000 to 4,000 pg g-1 (Korcak 1988). There are several mechanisms by which the high levels of manganese in the foliage may be detoxified. One hypothesis is that of compartmentalization (Foy er al. 1978). This is where ions are excluded from the active parts of the cell and concentrated in vacuolar and other inert regions as complexes or ions (Ernst 1975). There is further support for compartmentation as a 150 detoxifying mechanism. Memon and Yatazawa (1984) identified a manganese-oxalate complex in the supernatant fraction of leaf cells of the manganese accumulator Acanthopanax sciadophylloides. The supernatant constituted the vacuolar fraction and had the highest Mn concentration of all the fractions. Similarly, hemlock may tolerate high Mn by isolating it in vacuoles since, from the cellular fraction study, this fraction had the highest Mn level. The Mn may be in the free ionic state since most of the foliar Mn was water-soluble, or it may be weakly complexed judging from Memon and Yatazawa's findings (1982, 1984). C. Comparison and Consideration of Adequate Levels. i. Individual Nutrients According to vector analysis some of these hemlock stands had deficiencies of nutrients in addition to Zn and Mn (Figures 20 and 21). Although vector analysis indicates a strong Zn deficiency there were strong responses to other nutrients. It is interesting to consider these results in light of the laws of limiting factors. There are two laws which describe the relation of growth to limiting factors. Liebig's law of the minimum suggests growth is controlled by the most limiting factor. That is growth response to a nutrient will not occur unless deficiency of the most limiting nutreint is at first alleviated. This law is analogous to a barrel with staves of different sizes. The ability of the barrel to hold water is limited by the shortest 151 stave. This law may be limited to the situation in biology where the minimum factor is so low as to stop the process entirely (Daniel er al. 1979), perhaps where visual symptoms of a deficiency are evident. Mitscherlich's law of the minimum argues that increasing any factor that is below its optimum will improve growth, but increasing the factor furthest from its optimum will give the greatest increase in growth (Daniel et al. 1979). A response occurred to a "complete-Zn-Mn" treatment even though Zn was shown from vector analysis as being strongly limiting. This treatment was synergistic to Zn uptake. Therefore, it is difficult to say whether response was due to a nutrient in the "complete-Zn-Mn" treatment, the Zn, or an improvement in nutrient balance. According to the "barrel" analogy, a growth response should not have occurred in this study unless the Zn deficiency was first alleviated. This seems to demonstrate the limited applicability of the "barrel" approach in biological systems. It does not recognize that there are interactions between nutrients, such as synergisms or antagonisms, or a nutrient may improve growth creating a sink for another nutrient. The response data from the "complete-Zn-Mn" treatment (Table 13) were compared to existing foliar nutrient interpretations (Appendix I), to assess if the interpretations from the guidelines were consistent with the observed results. Ballard and Carter (1985) in their review (Appendix I) indicate an adequate foliar N threshold of 1.45 eg g-1. Both 152 sites had foliar V levels below 1.0 cg g""1 and 1.11 cg g-1 in the year of fertilization and they responded to 100 kg ha~x of N. This is in the severely deficient range. Fertilization brought foliar levels up to 1.67 cg g-t for both sites, which is adequate. Hardie (1985) found that 100 kg ha-1 of N and 150 kg ha-1 of P did not affect foliar N concentrations but total seedling dry mass was increased over the control. The control and treated foliar concentrations were both above the adequate threshold. Weetman et al. (1989) have also identified deficiencies of N and P in young regeneration of coastal western hemlock using foliar analysis. Deficiencies of N and P were confirmed by subsequent 3-year height growth response. Data from Gill and Lavender (1983) showed a response to 224 kg N ha-1, with control trees having foliar N levels of 1.33 to 1.53 cg g~x. Seedlings in the field having foliar N levels of 0.96 cg g-1 (severely deficient) responded to 200, 300 and 400 kg N ha-1 (Germain 1984). Zasoski and Gessel (1982) found that seedlings having a foliar N level of 0.88 cg g~x (very severely deficient) responded to application of 198 kg N ha-1. In a study of 940 plots of immature stands of Douglas-fir and hemlock in coastal B.C. measured over 12 years, 50 - 70% of hemlock stands responded to N fertilization. Fertilization*increased both total and merchantable volume but height was not significantly affected. Unthinned hemlock stands on fresh and moist sites responded to fertilization better than did those on the moderately dry sites (Omule 1990). 153 After the first year, site 4 had a control P level of 0.197 eg g-* and site 5 had 0.182 eg g-1. After the second year, site 4 had a foliar P level of 0.183 eg g-1- and site 5 had 0.167 eg g-1. When foliar P dropped to 0.167 eg g-x on site 5 after the second year, there was a nutrient response to P. When the foliar P level was higher there was no response. There have been reports of hemlock response to P fertilization. Hardie (1985) found increased P in seedlings fertilized with 150 kg P ha-1 with controls having a mean foliar P level of 0.124 eg g_1. Ballard and Carter (1985) suggest 0.15-0.35 eg g"~* represents the range of slightly deficient to adequate. Data from Heilman and Ekuan (1980) indicate that a P response to 300 kg of P ha-*- occurred to seedlings with foliar P levels in the severely deficient range (<0.11 eg g~M. Germain (1984) found that applications of P at either 50 kg ha-1 or 38 kg ha-1 increased foliar P levels of seedlings in the field which had control levels of 0.13 pg g-1. Zasoski and Gessel (1982) found that seedlings which had a foliar level of 0.08 eg g_1, which is considered deficient, responded to a P application of 447 kg ha-1. Adequate levels for foliar B are suggested to be in the range of 10-15 pg g""1 (Ballard and Carter 1985). An experiment by Majid (1984) with greenhouse grown lodgepole pine seedlings suggests a critical range for foliar B of 7 to 16 pg g_t. Control foliar B levels for sites 4 and 5 were 24.5 and 28.4 pg g-1- respectively, and B fertilization increased them to 33.2 and 45.8 pg g~x respectively. These are much greater than the 154 suggested critical range and the B response vectors (Figures 20 and 21) indicate a greater requirement for B. A foliar concentration of 4 pg g_l is proposed to be an adequate level for Cu. Field trials with lodgepole pine indicate a critical value for foliar Cu to be 4 pg g~x (Majid 1984). Foliar Cu on site 4 was 3.2 pg g-x and 3.1 for site 5. Fertilization increased foliar Cu on site 5 to 4 pg g-1 which is inferred to be just adequate with evidence of a deficiency from Figure 21A. Although Cu on site 4 may be classified as possibly inadequate there was no significant response. Foliar S concentrations were 0.16 and 0.12 eg g-x for sites 4 and 5 respectively, and the N/S ratio of control foliage on site 5 was 9.0. According to the diagnostic guidelines in Appendix I, site 4 is between a S deficiency unlikely and no S deficiency, there is no S deficiency, and a N induced deficiency is unlikely. The recommendations from the guidelines appear to be consistent with the results of the S treatments in which there was no increased uptake, suggesting that S is not limiting. There was no response to Ca, K, or Mg. Foliar Ca was 0.28 and 0.32 eg g-*- for sites 4 and 5, respectively, which is greater than 0.2 eg g-t critical value which is considered to be adequate. . Foliar K was 0.68 and 0.78 eg g~~x for sites 4 and 5 respectively. An adequate value for hemlock is suggested to be 0.8 eg g~x. However, since there was no increased nutrient 155 uptake, a lower value may perhaps be considered as adequate. Foliar Mg was 0.14 cg g-*- for both sites, which was sufficient compared to the level considered adequate of 0.12 cg g-1. Since there was no response to Ca, K or Mg fertilization, these limits considered adequate may be applicable for hemlock. In the case of K this limit may be reduced. The increase in foliar active Fe concentration on site 5 after the first year suggests that physiologically active iron is limiting. Foliar active Fe went from 26.2 pg g-1 for the control to 34.8 for the treatment. The guidelines suggest that 30 pg g-1 separates the "deficiency likely" category from the "deficiency unlikely" category. This seems to be a suitable recommendation for hemlock since an Fe nutrient concentration response occurred when foliar levels were below 30 pg g-1 on site 5, but there was no significant response where foliar levels where above 30 as on site 4. Foliar Fe levels were 25 and 39 pg g-1 for sites 4 and 5 respectively which are in the range of possible deficiency (25-50 ug 8_1>. Control foliar Zn concentrations (Table 8) are below the level considered to be adequate: 15 pg g~x . Since positive growth responses occurred to Zn fertilization, the diagnostic norms for Zn appear to adequately describe Zn nutrition of hemlock. Further evidence of a Zn deficiency comes from the strong Zn response to the "complete-Zn-Mn" treatment (Figures 20 and 21) 156 The diagnostic norms for Mn need to be revised for hemlock. Although foliar Mn concentrations for the controls (Table 8) were higher than the "stated" adequate level of 25 pg g-1, growth response to Mn fertilization occurred. ii. Nutrient Balance Ingestad's nutrient ratios were calculated for the foliar nutrients from the "complete -Zn -Mn" and the control treatments for sites 4 and 5. These data are presented in Table 32 along with Ingestad's ratios considered to be optimum for growth (Ingestad 1979). The nutrient ratios for both the "complete -Zn-Mn" and control treatments tend to be in balance relative to the optimum ratios. The exceptions are Mn and Fe which may be considered to be out of balance with respect to N, with Mn being too high and Fe being too low. This may be an indication of an insufficient supply of Fe. The "complete -Zn -Mn" treatment tended to maintain the foliar nutrient balance; however, after the first year of treatment, fertilization tended to decrease the ratios of K, P, and Cu from the optimum ratios. Since there had been an increase in foliar P and Cu concentrations, the decrease to suboptimal ratios suggests that these levels are insufficient and further additions are required. 157 Table 32. Ingestad's foliar nutrient ratios for the complete -Zn -Mn and control treatments on sites 4 and 5 for the years 1986 and 1987. The optimum ratios are from Ingestad (1979). Zn Mn K Ca Mg P TS Site 4 1986 Complete 0.1 8 39.8 18.1 7.1 9.6 10.4 Control 0.087 14.3 68 27.7 14 19.7 16 Site 4 1987 Complete 0.09 10.3 57.5 26.1 11.7 17 Control 0.1 13.2 65.1 27.9 13.6 15.6 Site 5 1986 Complete 0.09 6.4 48.4 18.1 8.2 12.8 Control 0.1 11.7 69.7 28.7 12.6 16.3 Site 5 1987 Complete 0.11 7.3 65.2 25.5 11.3 18.3 Control 0.1 10.8 72.7 27.3 11.4 14.4 Table 28 (concluded). Fe Cu B AFe AZn ANn Site 4 1986 Complete 0.23 0.021 0.2 0.22 0.11 2.66 Control 0.25 0.032 0.25 0.32 0.068 12.4 Site 4 1987 Control 0.42 0.031 Complete 0.42 0.031 Site 5 1986 Complete 6.8 0.28 0.024 0.27 0.21 Control 11.1 0.35 0.028 0.26 0.24 Site 5 1987 Complete 0.41 0.077 Control 0.46 0.034 Ingestad's Ratio Zn Hn K Ca Mg P 0.03 0.4 70 8 5 16 TS Fe Cu B 9 7 0.03 0.2 159 Since there had been no increase in the foliar concentration of R but uptake had been maintained and there was a growth response, these results could suggest that the true optimum ratio for K is actually not so high. D. Retranslocation The existence of nutrient deficiencies may be assessed by determining the occurrence and the extent of nutrient remobilization. Remobilization of mineral nutrients is important during ontogenesis in periods of insufficient supply of nutrients to the roots during vegetative growth (Kramer and Kozlowski 1979). If a plant is under a nutrient stress some nutrients will be remobilized from the old foliage and translocated to the new foliage. This retranslocation could then be used as a means of diagnosing the nutrient status of a plant. In the second year following soil applications of Mn the current year's foliage and two-year-old foliage continued to accumulate Mn. Therefore, it could not be determined if retranslocation occurred. Retranslocation of Zn occurred in the second year following fertilization from the the high foliar Zn treatment. This result was in contrast to the situation in P. radiata found by McGrath and Robson (1984). In their seedlings, the Zn concentration of the older primary needles remained constant regardless of the Zn status of the seedlings. In contrast, the results from this research for Zn are consistent 160 with the finding that remobilization of Zn from old leaves depends upon the Zn status of the plant. Zn is more mobile when the concentration is high (Kramer and Kozlowski 1979). For example, subterranean clover plants given luxury supplies of Zn moved up to 25% of the Zn in their old leaves and petioles into developing fruits, whereas those given deficient or marginal supplies moved little or none at all (Loneragan 1976). There is evidence that the decrease in foliar Zn concentrations in the two-year-old foliage was due to remobilization. Firstly, foliar Zn concentrations in the control foliage did not decrease with foliar age. Secondly, Zn has been found to be leached with difficulty. Less than 1% of the Zn was leached with water applied for 24 hours to young leaves of squash and beans (Tukey et al. 1958). E. Nutrient Interactions Applications of Zn had no effect on uptake of Mn and in some cases applications of Hn increased foliar Zn. In soybeans, Zn has been shown to increase the translocation of Hn to the tops of the plants, which can lead to Hn toxicity (Foy 1983). However, an inverse relationship was found between Zn application and Hn in soybean (White er al. 1979), maize (Singh and Steenberg 1974), and bush beans (Ruano er al. 1987). 161 Since there appeared to be no antagonism between foliar Zn concentrations and Mn applications or foliar Mn concentrations and Zn applications, the evidence from this study indicates that it is unlikely the lower foliar Zn levels in these hemlock stands are due to Mn antagonism. The evidence from the literature suggests an antagonism between Zn and Mn (Reddy et al. 1978; Nair et al. 1977; Malavolta er al. 1956; Hawf er al. 1967). However, in agreement with the results in this study, Kohno er al. (1984) found in bean plants and Shuman and Anderson (1976) found in soybeans that the foliar levels of Zn increased with increasing Mn concentrations in the solution. It is interesting that soil applications of Zn did not increase foliar Zn after the first year but in some cases soil applications of Mn did. Since there was a positive growth response with Mn in some cases this may have created a sink for Zn. This may suggest an increased Zn requirement with Mn. With similar soil application levels of Zn and Mn (200 kg ha-1) a greater percentage of Mn was found in the foliage compared to the Zn treatment. This suggests different soil chemical reactions or plant uptake/translocation mechanisms for the two elements. Different retention and/or release mechanisms of the soil for Zn and Mn may be ruled out because hemlock had different foliar levels of Zn and Mn in comparison to other tree species in the same stands. This would indicate that the differences between foliar levels of the two elements is due to 162 plant factors such as differences in nutrient uptake or in translocation. Nair and Prabhat (1977), found that with identical application rates, relatively more Zn was root absorbed than Mn, but of the amount root absorbed, relatively more Mn was translocated to the shoots. This would suggest different mechanisms of translocation for Zn and Mn. An interaction was found between foliar Zn and foliar N concentrations. An interaction between Zn and N in trees has been found in several other studies. Results of Zasoski and Porada (1986) indicate a strong relationship between N and Zn in hemlock growing on slashburnt sites. Foliar levels were <1.1 cg g_l N and <12 pg g-i Zn, which according to a DRIS analysis were strongly growth limiting. High applications of N did not stimulate growth nor increase foliar N levels, which suggested that Zn was inadequate for N. McGrath and Robson (1984) investigated the effect of N and P supply on the response of P. radiata to Zn. They found that a response to Zn depended on the requirements for N and P being provided. Evidence from these empirical studies implies an interaction between Zn and N. There is evidence in the literature to support a functional relationship between Zn and N. Firstly, this interaction may occur because of the effect of one element on the transport of the other element in the soil or the plant. For example transition metals such as Zn have high affinities for -M-ligands (Robson and Pitman 1983). 163 The second way in which this interaction may occur is in protein synthesis. There are three mechanisms by which Zn affects protein synthesis. Nitrogen is known to be an essential component of proteins. Zinc deficiency causes a reduction in protein and ribosomal RNA contents (Kitagishi and Obata 1986. 1987; Obata and Umebayashi 1988; Prask and Plocke 1971; Wacker 1962; Sharam er al. 1981). Concurrently there is an accumulation of amino acids and amides (Kitagishi and Obata 1986; Wacker 1962; Possingham 1956). The first mechanism involves the transcription of DNA by RNA. Zinc forms chemical bonds with the amino acids cystine and histidine in such a way that the chain of amino acids becomes folded around the zinc to form a loop or 'zinc finger* (Figure 27). Proteins (RNA polymerase) that regulate the transcription of DNA to RNA do so through their zinc fingers. If the zinc is absent, the protein cannot bind to DNA and regulate its transcription (Parraga er al. 1988). Therefore, zinc is essential in terms of its structural role. The second way is through the occurrence of Zn in the total RNA fraction, and its effect on the ribosomes; Zn may be required to maintain the structural intergrity of the ribosomes (Prask and Plocke 1971). 164 Figure 27. Ribbon model of a single zinc finger domain (ADRla) incorporating tetrahedral coordination of zinc by cystine (C) and histidine (H) (from Parraga et al. (1988)). 165 The third mechanism involves the regulation of RNA degradation by Zn. Higher rates of RNAase activity are observed with Zn deficiency (Marschner 1986). This demonstates the importance of Zn for protein synthesis. F. Foliar Application of Zn in Forestry Foliar-applied sprays are the most efficient means of supplying hemlock trees with Zn. This method would appear to be of impractical use in larger fertilization experiments or in operational fertilization of stands. However, pesticides in solution form are routinely applied in operational forestry with fixed-winged aircraft and helicopters. This same technology could be applied to the foliar application of nutrients. 166 CHAPTER 6. CONCLUSIONS This study was undertaken in order to investigate the the significance of the characteristic pattern of foliar Zn and Mn concentrations of hemlock in its nutrition. Two approaches to the study were used: comparative nutrition and the screening trial-growth response technique. In a comparison of total foliar concentrations, hemlock had lower Zn compared to Douglas-fir, amabilis fir and white pine. In contrast, hemlock had higher Mn compared to Douglas-fir, amabilis fir, white pine, red cedar and yellow cedar. Analysis of cellular fractions of foliage produced two results. First, zinc and manganese accumulated in two different fractions irrespective of the level of the treatment or the species. Accumulation in certain fractions may indicate a physiological need in that fraction or a tolerance mechanism. Second, comparing hemlock to Douglas-fir total Zn levels tend to be consistent with levels in different fractions, indicating total levels may be an adequate indication of physiological levels. Comparing hemlock to Douglas-fir, total Mn levels are consistent with Mn levels in different fractions, indicating hemlock may have a greater physiological Mn requirement or a Mn tolerance mechanism. 167 There were both a nutrient concentration response and a growth response to zinc applications. The timing of response was dependent upon the method of application. Response to foliar applications occurred in the first year following treatment, and response to soil applications occurred in the second year following treatment. Growth response as measured by shoot increment ratio was obtained primarily in the second year after treatment to foliar applications of zinc. Height increment ratio increased in response to foliar zinc applications in the second year. The fact that there were cases of positive growth responses to Zn fertilization is evidence that the relatively lower foliar Zn concentrations of hemlock compared to some other conifers do indicate some deficiency of Zn in the stands used for this study. Additional evidence of a Zn deficiency are; the higher foliar Zn levels associated with optimum height increment compared to control foliar Zn levels, retranslocation of Zn in the second year following the high foliar Zn treatment and the high ranking of Zn in the vector graphs from the "complete-Zn-Mn" treatment. In some cases, rather than a growth response to Zn, luxury consumption of Zn occurred. For plants having low nutrient requirements, luxury consumption is a physio-ecological mechanism which supplies the plant during periods of nutrient insufficiency between the pulses of luxury availability. A similar situation 168 may operate for hemlock, since this species has low nutrient requirements. There were both positive nutrient and growth responses to soil Hn treatments. Positive nutrient concentration and growth responses occurred in the first year. Foliar Hn was still elevated in the second year in the one-year-old and two-year-old foliage. The elevated foliar Hn in the second year must have been from increased uptake from the soil or translocation from the roots, since there was no retranslocation from the two-year-old foliage. Hemlock has foliar Hn levels which are considered toxic in some plants, and the fact that hemlock continued to take up soil-applied Hn with positive growth responses indicate that hemlock is Hn-tolerant and also has increased requirements for Hn. The foliar Hn levels may not adequately reflect the physiological requirements for Hn. There are both a physiological component and an ecological component of a plant's nutrition. For Hn, there may be two physiological pathways in hemlock. One pathway may be active in complexing the metal, operating as a tolerance mechanism, giving hemlock a competitive advantage in acid soil. The second pathway may channel the metal into the growth metabolism of the plant. How the allocation of manganese is regulated between the two competing paths is not known. In general this situation may apply to metal-tolerant plants where a metal on the one hand is being detoxified by the 169 tolerance mechanism, but on the other hand is required in growth metabolism. There must be a mechanism which regulates the allocation of the metal between the two pathways. This topic which involves both ecological and physiological plant nutrition requires greater investigation. Determining the nutrient status of a micronutrient in a plant which is an accumulator of the micronutrient may be problematic. It is necessary to determine the physiologically active fraction of the nutrient. This may be done using extracts of the nutrient whose level is correlated to the rate of a physiological activity, or separation and analysis of cellular fractions which are involved in a physiological process. Foliar application of Zn and soil application of Mn appeared to be the most efficient means of supplying the plant with these nutrients. Increased foliar levels of Zn did occur with soil Zn treatments but this was delayed until the second year after fertilization. It is hypothesized that this is due to inhibited uptake and/or translocation of Zn. Low foliar Zn concentrations in hemlock are not due to low inherent zinc fertility of the soil, since species which coexist with hemlock in the same stands tend to have higher foliar zinc. A growth response (foliar mass per shoot and shoot increment ratio) was obtained with the "complete-Zn-Mn" treatment. Vector analysis which ranked the relative responses 170 revealed the existence of nutrient deficiencies other than Zn and Mn. Vector analysis also revealed evidence of a strong Zn deficiency. Foliar Zn was synergistic to the "complete-Zn-Mn" treatment in both the first and second years after treatment. Therefore, it was difficult to say whether the growth response was due to the applied nutrients, the Zn, or an improved balance of all nutrients. Conceivably, fertilization could lead to a Zn deficiency if the native supply in the soil cannot meet the increased demand by the plant. Such a situation is termed an induced deficiency. Induced Zn deficiency or other nutrient deficiencies may be additional reasons why growth response in hemlock to nitrogen fertilization has been inconsistent. Ingestad's nutrient ratios were calculated for the foliar levels from the control and the "complete-Zn-Mn" treatments. Comparing these ratios to the optimum revealed that most of the nutrients were in balance except for iron and manganese. Existing diagnostic norms for Zn appear to adequately describe the Zn nutrition of hemlock. Response to fertilization occurred with control foliar Zn concentrations for hemlock being below the critical level of 15 pg g~x. Diagnostic norms for Mn need to be revised. Response occurred even though control foliar Mn concentrations for hemlock were well above the critical level of 25 pg g-1. Therefore, total foliar Mn may not be indicative of the physiological Mn status of hemlock. 171 The empirical relationship found between foliar Zn and N suggests an interaction between the two elements. The response of the plant to the addition of one of these elements is dependent upon the adequate supply of the other element. This interaction requires further investigation to determine if variability in response to N fertilization may be affected by variability in the Zn status of the plant. Foliar Zn was rather than being antagonistic was in some cases synergistic to soil applications of Mn . Therefore, there was no evidence in this study to suggest that low foliar levels of Zn in hemlock are due to a Mn antagonism. The only interaction obtained with the "complete-Zn -Mn" treatment was a synergism with foliar Zn. There are several aspects of this research which require further.investigation. For the information from these screening trials to be applied to the management of hemlock, several further steps need to be taken. The next step following the analysis of screening trials would be studies of correlation between site factors and responsiveness. This information would then be used for classifying operational stands into response categories. Another aspect which needs investigating is the interaction between nitrogen nutrition and zinc. Since there is evidence of deficiencies of Zn, Mn and other micronutrients optimum nutrition experiments need to include these. This may be done by supplying optimum dosages of macronutrients wit'h 172 different dosages of individual micronutrients or different balances of micronutrients. Manganese nutrition of hemlock may be similar to that of other metal-accumulating plants in that there is a high requirement for the metal. This would involve investigation of the regulation of Mn between the two physiological pathways of metal-tolerance and growth. Luxury consumption may be a characteristic of plants having low nutrient requirements. Luxury uptake of nutrients would occur during periods of high nutrient availability and utilization during periods of low nutrient availability. A further study of the nutritional significance of luxury consumption of nutrients would be of interest. 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Municipal sewage sludge use in forests of the Pacific Northwest, U.S.A.: Environmental Concerns. Waste. Manage. Res., 2:227-246. 188 APPENDIX A. SITE AND SOIL DESCRIPTION APPENDIX Al. SITE I NTS Sheet: Port Coquitlam 92/G7 Location: UBC Research Forest Road: F70 Long: 122* 34' 10" Lat: 49* 17' 40" Elevation: 360 m Slope: Very gentle slopes 2-5% Aspect: North-west Landform: Till over bedrock Parent Material: Till Drainage Class: Well Perviousness: Moderate Depth of Pit: 52 cm Rooting: 52 cm Depth to Water Table: Never Present Compact Till: 45 cm Bedrock: 52 cm Soil Classification: Orthic Humo-Ferric Podzol Humus Form: Humimor BGCZ: Windward Submontane Maritime Coastal Western Hemlock Wetter CWHbl Major Vegetation: Tsuga beterophylla, Abies amabilis, Pseudotsuga menziesii (planted) Lower Vegetation: Vaccinium parvifolium, Hylocomium splendens, Vaccinium alaskaense, Blechnum spi cant. 189 Soil Profile LFH 3-0 Black (10 YR 3/1, moist); fresh and partially decomposed organic matter; clear and irregualr boundary, 3-5 cm thick. Bf 0-42 Dark yellowish brown (10 YR 4/4, moist); loamy sand; single grained; fine sub-angular blocky; slightly sticky, slightly plastic, very friable; few fine and coarse roots; about 40% coarse fragments; abrupt, irregular boundary; C 42-52 Brown (10 YR 5/3, moist); loamy sand; singled grained; fine-coarse sub-angular blocky; slightly sticky, slightly plastic, very firm; very few medium and coarse roots; about 40% coarse fragment content; abrupt, irregular boundary; 190 APPENDIX A2. SITE 2 NTS Sheet Location: Road: Long: Lat: Elevation Slope: Aspect: None Landform: Till Parent Material Drainage Class: Perviousness: Depth of Pit: Rooting: Depth to Water Port Coquitlam 92/G7 UBC Research Forest E12 122° 34' 10" 49° 18' 50" 440 m Very gentle slopes 2-5% over bedrock : Till Imperfectly Drained Slowly pervious 43 cm 43 cm Table: Never Present Compact Till: None Bedrock: 43 cm Soil Classification: Rego Gleysol Humus Form: Humimor BGCZ: Windward Submontane Maritime Coastal Western Hemlock Wetter CWHbl Major Vegetation: Tsuga heterophylla, monticola Lower Vegetation: Vaccinium parvifolium, NU yl. i if i ti litil.phu n lorn u Thuja plicata, Pinus Gaultheria Ki. nit ft if r,if J. n shallon, oregana 191 Soil Profile LFH 15-0 Black (10 YR 3/1, moist); fresh and partially decomposed organic matter; abundant very fine and fine, and few medium roots; abrupy, wavy boundary; 15-20 cm thick. Cg 0-18 Gray (10 YR 5/1, moist); loamy sand; sinle grained; moderatley, fine, subangular blocky; slightly sticky, slightly plastic, very friable, soft; plentiful very fine and fine roots; about 30% coarse fragments; abrupt and wavy boundary; 7-20 cm thick. APPENDIX A3. SITE 3 NTS Sheet: Chilliwack 92/H4 Location: Chipmunk Creek Road: Chipmunk.creek Road Long: 121° 41' 50" Lat: 49° 8' 10" Elevation: 880-960 m Slope: Very strong slopes 31-45% Aspect: South-east Landform: Till over bedrock Parent Material: Till Drainage Class: Rapidly Drained Perviousness: Rapidly pervious Depth of Pit: 1.5 m Rooting: 60 cm Depth to Water Table: Never Present Compact Till: 60 cm Bedrock: None Soil Classification: Orthic Humo-Ferric Pod2ol Humus Form: Humimor BGCZ: Windward Montane Maritime Coastal Western Hemlock Wetter CWHb2 Major Vegetation: Tsuga heteropbylla, Abies amabilis, Pseudotsuga menziesii (planted) Lower Vegetation: Rhytidiopsis robusta, Vaccinium alaskaense, Blecbnum spicant, Moneses uniflora 193 Appendix A3 (continued) Soil Profile L 2-0 Black (10 YR 3/1, moist); fresh and partially decomposed organic material; abundant fine and very fine roots; abrupt, broken boundary. Ae 0-5 Gray (10 YR 6/1, moist); loamy sand; single grained; weak, fine-coarse subangular blocky; nonsticky, nonplastic, loose, soft; abundant very fine-fine roots; about 5% coarse fragments; abrupt, broken boundary; 0-5 cm thick. Bf 5-60 Yellowish Brown (10 YR 5/6, moist); loamy sand; single grained; moderately, fine-coarse subangular blocky; slightly sticky, slightly plastic, very friable, soft; abundant fine-medium, and very few coarse roots; about 30% coarse fragment content; gradual wavy boundary; 45-60 cm thick. Cg 60-*- Gray (10 YR 6/1, moist); coarse sand; single grained; stong, fine-coarse subangular blocky; slightly sticky, slightly plastic, very friable, soft; about 50% coarse fragment content. 194 APPENDIX A4. SITE 4 NTS Sheet: Port Coquitlam 92/G7 Location: UBC Research Forest Road: E13 Long: 122° 34' 20" Lat: 49° 18' 50" Elevation: 520 m Slope: Nearly level 0.5-2% Aspect: North Landform: Till over bedrock Parent Material: Till Drainage Class: Well Drained Perviousness: Moderately pervious Depth of Pit: 90 cm Rooting: 70 cm Depth to Water Table: Never Present Compact Till: 80 cm Bedrock: None Soil Classification: Duric Humo-Ferric Podzol Humus Form: Humimor BGCZ: Windward Submontane Maritime Coastal Western Hemlock Wetter CWHbl Major Vegetation: Tsuga heterophylla, Abies amabilis, Pseudotsuga menziesii (planted) Lower Vegetation: Vaccinium parvifolium, Gaultheria shallon, Rhytidiadelphus loreus, Kindbergia oregana 195 Soil Profile LFH 20-0 Black (10 YR 3/1, moist); freash, partially and well decomposed organic material; abundant fine and very fine, and few coarse roots; gradual smooth boundary; 5-20 cm thick. Ae 0-10 Gray (10 YR 6/1, moist); loamy sand; single grained; moderately, medium, subangular blocky; slightly sticky, slightly plastic, very friable, soft; abundant fine and medium roots; about 30% coarse fragments; clear and irregular boundary; 0-10 cm thick. Bf 10-46 Dark yellowish brown (10 YR 4/4, moist); loamy sand; single grained; moderately, medium, subangular blocky; slightly sticky, slightly plastic, very friable, soft; few fine and medium roots; about 30% coarse fragment content; clear and wavy boundary; 36 cm thick BCc 46-72 Yellowish red (5 YR 5/8, moist); loamy sand; single grained; moderately, medium, subangular blocky; slightly sticky, slightly plastic, very friable, soft; about 30% coarse fragment content; clear and wavy boundary; 10-26 cm thick; C 72+ Yellowish brown (10 YR 5/6, moist); coase sand; single grained; moderately, medium, subangular blocky to platy; slightly sticky, slightly plastic, very friable, soft; about 50% coarse fragment content. APPENDIX A5. SITE 5 NTS Sheet: Stave Lake 93/G6 Location: Mission Tree Farm Road: Rock Creek Road Long: 122° 34' 20" Lat: 49° 18' 50" Elevation: 1,100-1,200 m Slope: - Very Strong Slopes 31-45% Aspect: East Landform: Till over bedrock Parent Material: Till Drainage Class: Well Drained Perviousness: Moderately pervious Depth of Pit: 80 cm Rooting: 40 cm Depth to Water Table: Never Present Compact Till: 80 cm Bedrock: None Soil Classification: Orthic Ferro-Humic Podzol Humus Form: Humimor BGCZ: Windward Montane Maritime Coastal Western Hemlock Wetter CWHb2 Major Vegetation: Tsuga heterophylla, Abies amabilis, Chamaecyparis nootkatensis, Pseudotsuga menziesii (planted) Lower Vegetation: Vaccinium alaskaense, Dryopteris expansa, Blecbnum spicant, Sphagnum gicgensohnd-i, Tiarclla uni foliata 197 Appendix AS (continud) Soil Profile LFH 25-0 Black (10 YR 3/1, moist); fresh, partially, and well decompose organic material; abundant very fine and fine, few coarse, and plentiful medium; abrupt and smooth boundary; 20-25 cm thick. Bhf 0-18 Black (10 YR 3/1, moist); loamy sand; single particles; strong, fine-very coarse subangular blocky; sticky, plastic, very firm, soft; plentiful fine, and medium roots; about 20% coarse fragment content; abrupt, smooth boundary; 18-20 cm thick. Bf 18-35 Dark yellowish brown (10 YR 4/4, moist); loamy sand; single particles; strong, fine-very coarse subangular blocky; sticky, plastic, very firm, soft; very few fine, and medium roots; about 402 coarse fragment content; abrupt, smooth boundary; 17-20 cm thick. C 35+ Light gray (7.5 YR 7/0, moist); sand; single grained; red (2.5 YR 4/8, moist) mottles; nonsticky, nonplastic, very friable, soft; about 50% coarse fragment content. 198 APPENDIX Bl. MODIFIED PARKINSON AND ALLEN DIGESTION FOR PLANT TISSUE ANALYSIS 1) Weigh 1 g (to the nearest mg) subsample of oven-dried (70°C for 3 hours), ground foliage and place in 100 ml digestion tube. 45 tubes in each set can be prepared, with a reference sample and a blank in each set. 2) Add 5 ml of cone. HaSO* (reagent grade) to each sample, and mix on a mechanical vibrator immediately. 3) Dispense 1 ml of LiaSO* - HaOa mixture (prepared by mixing 7.0 g L12S0A, 0.21 g selenium powder in 175 ml 30% HaOa) into each tube. Wait until 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 discontinuous heating to overcome initial foaming; that is, 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 LiaSO* - HaOa mixture to each tube. Wait till 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 HaOa 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 a final volume (100 ml) with demineralized water. 13) Cover tubes with an inert stopper, invert 3-4 times to mix, and pour contents into a labelled 125 ml plastic bottle. 199 APPENDIX B2. NITRIC ACID DIGESTION FOR ANALYSIS OF COPPER AND IRON IN FOLIAGE 1) Weigh 0.7 g (to the nearest mg) subsample of oven-dried (70°C for 3 hours), ground foliage and place sample into digestion tube. A set of 45 tubes can be prepared for one run, each set having a reference sample and a blank. 2) Add 5 ml cone. HNOa to sample, and mix on mechanical vibrator. 3) Cover the tubes with glass marbles and heat on digestion block at 40°C for 1 hour. 4) Increase heat up to 140°C and continue heating for 2 hours, couting from time the block reaches 140*0. 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 an inert rubber stopper and mix content by inverting 3-4 times. Transfer contents to a 60 ml plastic bottle. 7) Analyze the solutions for iron and copper by atomic absorption spectrophotometry. . 200 APPENDIX B3. PROCEDURE FOR THE DETERMINATION OF SULPHATE-SULPHUR IN FOLIAGE Extraction from foliage 1) Weigh one gram of oven-dried foliage into an erlenmeyer flask. Add 20 ml of 1 N HCL and record weight. 2) Heat on a hot plate to boiling, then continue boiling for 10 minutes. 3) Add water to bring back to the original weight. 4) Filter through #42 filter paper. Digestion 1) Use from 0.5 to 2 ml of sample. 2) Place 5 ml of 1 N NaOH into a 20 ml test tube and attach so the delivery tube reaches almost to the bottom of the test-tube. 3) Add 4 ml of reducing agent (mix 300 ml of hydriodic acid (57% and 1% preservative) with 75 ml of 50% hypophosphorous acid and 150 ml of 90% formic acid. Boil gently with a stream of Na flowing through the solution for 10 minutes after the temperature reaches 115°C. Cool with the Na still flowing) and sample up to 40 pg S in an aliquot to the boiling flask. Adjust the N2 flow to 75 ml minute-1 per flask. 4) Heat for 20 minutes so it is just barely boiling. 5) Remove, and add 2.5 ml bismuth reagent and mix. Read immediately at 400 mp and compare against a standard. APPENDIX B4. DETERMINATION OF ACTIVE IRON IN FOLIAGE 1) Weigh 0.5 g subsample of oven-dried (70°C for 3 hours), ground foliage into a 60 ml screw-capped plastic bottle. 2) Add 10 ml 1 N HCL (reagent grade) in demineralized water to each sample. Tightly cap the bottle to prevent leakage. (70 samples can 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) Filter the extract through Whatman # 41 filter paper and collect the filtrate in a 60 ml plastic bottle. 5) Analyze for iron on atomic absorption spectrophotometer. Analysis should be done within 48 hours. 202 APPENDIX B5. DETERMINATION OF EXTRACTABLE ZINC 1) Weight 0.5 grains of a subsample of oven-dried (70°C for 3 hours), ground sample into a 60 ml plastic bottle. 2) Add 20 ml of 1.0 mM MES (2-(N-morpholino)ethanesulfonic acid) (prepared in demineralized water) to each bottle. 3) Shake for 5 hours horizontally on a reciprocating shaker at room temperature. Include a blank and a reference sample with each set. 4) Filter through Whatman #41 filter paper. 5) Analyze for Zn on atomic absorption. 203 APPENDIX B6. WATER EXTRACTABLE MANGANESE FROM FOLIAGE 1) Weigh out 0.1 g of a subsample of oven-dried (at 70°C for 3 hours), ground foliage into a 60 ml plastic bottle. 2) Add 50 ml of demineralized water. 3) Shake for 1 hour horizontally on a reciprocating shaker at room temperature. Have a blank and reference sample for each set. 4) Filter through Whatman #41 filter paper. 5) Determine Mn using atomic absorption on the extracts. 204 APPENDIX C. COMPARISON OF FOLIAR Zn, Mn, AND Fe LEVELS USING AA VERSUS ICP SAMPLE ZnAA ZnlCP MnAA MnlCP FeAA FelCP Ug g"1  Given 1 30 21.7 65 82 .6 170 245. 2 28 22 .2 66 83.3 170 252 3 29 33.9 180 200 75 79.7 4 40 33.8 220 200 70 82 .8 5 58 53.5 240 207 220 361 6 58 53.2 240 210 260 315 7 13 6.8 620 633 35 7.6 8 185 178.6 1220 1344 50 16 .9 9 23 15 .6 540 510 30 12.5 10 22 14.9 520 507 35 14.4 11 27 21.3 2400 2506 35 0.5 12 60 58 1420 1521 32 5.2 13 113 107.6 880 846 30 7.3 14 114 107.5 840 845 32 12 15 18 9.9 1200 1198 35 15 .4 16 18 10.7 1100 1164 40 19.5 17 17 8.8 1080 1092 33 4.2 18 15 8.9 3880 3783 30 0.5 19 19 12.6 3120 3192 30 0.5 20 19 12.3 3100 3182 27 0.5 21 15 9.7 1040 1064 31 10.2 22 16 9.9 960 1076 32 14.2 23 11 5.6 600 583 32 9.9 24 23 16.3 900 937 30 7.2 25 24 16 .4 860 946 30 6.1 26 28 21.2 64 79.4 160 227 27 38 30.5 200 184 75 81 . 7 28 55 50.3 20 201 255 37 Sample Zn Mn Fe NBS 25 91 300 Tomato 62 238 690 1. Sample Numbers 1. 2, 26 National Bureau of Standards (NBS) Orchard Leaves 3, 4, 27 Pine Reference 84-3 5, 6, 28 NBS Tomato Leaves 8-25 Hemlock Foliage Samples 2. (U.S. Dept. of Commerce 1977) 3. (U.S. Dept. of Commerce 1976) 205 APPENDIX D. FORMULAS TO CONVERT AA VALUES TO THE CORRESPONDING ICP VALUES1. Equation for Zn Equation for Mn y = 1.005x Ra = 0.996 SE » 2.53 y = 1.017x Ra = 0.998 SE = 63.57 - 6.075 Where x = the concentration measured using the AA y = the equivalent concentration on the ICP Data are from Appendix C. 206 APPENDIX E. RECOVERY OF ELEMENTS IN NATIONAL BUREAU OF STANDARDS SAMPLES USING ICP AND AA CalCP CaAA MglCP MgAA KICP KAA •eg g ORCHARD LEAVES 1.98 1.47 0.605 0.566 1.4 1.42 1.88 1.37 0.616 0.566 1.42 1.36 1.89 1.4 0.577 0.574 1.39 1.37 1.92 1.41 0.599 0.569 1.4 1.38 MEAN GIVEN 2.09 2.09 0.62 0.62 1.47 1.47 % RECOVERY 91.9 67.5 96.6 91.8 95.2 93.9 TOMATO LEAVES 2.94 2.05 0.644 0.602 4.18 4.2 2.87 2.18 0.653 0.61 4.18 3.92 2.7 2.01 0.625 0.604 4.05 3.84 MEAN GIVEN X RECOVERY 2.84 2.08 0.641 0.605 4.14 3.99 3 3 0.7 0.7 4.46 4.46 94.7 94.7 91.6 86.4 92.8 89.5 207 Appendix MnlCP E (concluded) MnAA ZnlCP ZnAA FelCP Fe AA A1ICP ug g ORCHARD LEAVES 82.6 65 21.7 30 245 170 241 83 .3 66 22 .2 28 252 170 233 79.4 64 21.2 28 22 7 160 218 MEAN 81.8 65 21.7 28.7 241 167 231 GIVEN 91 91 25 25 300 300 X RECOVERY 89.9 71.4 86.8 114.8 80.3 55 .7 TOMATO LEAVES 207 240 53.5 58 361 220 371 210 240 53.2 58 315 260 315 201 200 50 .3 55 370 255 389 MEAN 206 227 52.3 57 349 184 358 GIVEN 238 238 62 62 690 690 Al AA 180 180 160 173 240 290 255 262 % RECOVERY 86.6 95.4 84.4 91.9 50.6 26.7 208 APPENDIX F. PREPARATION AND ANALYSIS OF CELLULAR FRACTIONS FROM FOLIAGE 1) Weigh 20 g of oven-dried (70°C for 3 hours), ground sample of foliage into 500 ml plastic bottles for the centrifuge, and add 100 ml of a sucrose-buffer solution (0.5 M sucrose, 0.05 M Tris-HCL). Shake to wet the plant material thoroughly. 2) A progressive separation was done using centrifugation; Fraction A 500 g for 5 minutes Fraction B 3000 g for 10 minutes Fraction C Settled by gravity Fraction D 15000 g for 30 minutes Fraction E Supernatant remaining The fraction was the pellet formed in the bottom of the centrifuge tube following centrifugation of the supernatant. The remaining supernatant was decanted and recentrifuged at the next highest speed. Fraction C formed following decanting the supernatant from which fraction B had been formed and before centrifugation at 15000 g. 3) Fractions A to D were dried in an oven in porcelain crucibles to a constant weight. Fraction E was stored in 60 ml plastic bottles in the refrigerator. 4) Fractions A to D were digested using the Parkinson and Allen acid digestion. Because of the small amount of material from fraction D, the acid used for the digestion was added to the crucibles left overnight, and then transferred to the digestion tubes. 5) Zn and Mn were determined on the digests and on the fraction E supernatant using atomic absortion spectrophotometry. 209 APPENDIX G. FIXATION AND EMBEDDING PROCEDURE 1) Place tissue in a small vial to completely cover it containing 2.5% glutarnaldehyde in 0.1 M Na-cacodylate (pH 7.2-7.4). Leave it for 3 hours. 2) The solution is prepared by combining 10 ml of 25% glutaraldehyde, 50 ml of 0.2 M Na-cacodylate and 40 ml of demineralized water. 3) The tissue is than rinsed three times at 10-minute intervals in 0.1 M Na-cacodylate (pH 7.2-7.4). This is prepared by adding 40 ml of 0.2 M Na-cacodylate to 40 ml of demineralized water. 4) The tissue is fixed for a second time for 1 hour in a solution of 1% osmium tetroxide in 0.1 M Na cacodylate (pH 7.2-7.4) . Thi.!i is prepared by adding a 1:1:2 volume of 2% aqua osmium t« t roxt d« s <lc*t« i n« r*l i y,*t<\ ««t ** r HIHI Na 5) The tissue is rinsed in demineralized water three times at 10 minute intervals. 6) The tissue is then taken through a dehydration series using ethanol at 10-minute intervals in the following concentrations of ethanol; 30%, 50%, 70%, 85%, 95%, 100%, 100%. 7) The tissue is then embedded in the following series; for 10 minutes in propylene oxide, twice, for 3 hours in 3:1 mixture of propylene oxide and plastic, for 7-12 hours in 1:1 propylene oxide and plastic, for 7-12 hours in 1:3 propylene oxide and plastic, and then 7-12 hours in 100% plastic. The tissue is then polymerized. 8) The plastic is prepared by mixing 34.58 g of Epon Ep812 WPE #190, 13.0 g of DDSA (dodecenyl succinic anhydride, 13.0 g of NMA nadic methyl anhydride, and 0.75 g of DMP -30. 210 APPENDIX H. THE MEHLICH 3 SOIL EXTRACTION METHOD 1) Weigh 5.0 g of soil samples into 125 ml plastic bottles. Include 2 blanks and 2 references soils per run. 2) Have extracting solution (M3) in 4 L carboy with outlet connected to teflon tubing closed with clip. Set carboy on stool on work bench. 3) Calibrate plastic graduated cylinder "to deliver" 50 ml. 4) Set up required number of 60 ml plastic bottles with funnels and Whatman #541 X 15.0 cm filter paper. 5) Since extraction time of samples should be precise use a stopwatch on the bench. 6) Fill graduated cylinder with M3 solution to the "to deliver" mark. 7) Pour into 1st 125 ml bottle, cap and put onto shaker at slow speed, starting the stopwatch. 8) Refill the graduated cylinder and, when stopwatch gets to 50 seconds, pour M3 solution into a 2nd 125 ml bottle, cap and put onto shaker. 9) Continue this at every 50 second reading (i.e. at 60 second intervals) of the stopwatch until the 6th bottle is put on the shaker, then immediately remove the 1st bottle (which will have shaken for 5 minutes) and pour through filter. Then transfer the label to the 60 ml collection bottle. 10) Continue adding 50 ml M3 solution to samples in 125 ml bottles at 50 second readings on the stopwatch, putting the bottle immediately onto the shaker, and then immediately removing and filtering the sample which has shaken for 5 minutes. Thus there should always be 5 bottles shaking after the initial start up, until the last 5 samples, which should be removed from the shaker at 1 minute intervals, starting at the 58 second reading on the stopwatch. 11) Measure elements on ICP. 12) Extracts may be kept for a week, however dilutions must be read within 3 days. 211 Appendix H (concluded). Extracting Solution Reagents: All should be ACS grade. Rl Ammonium nitrate R2 Ammonium fluoride R3 Acetic acid, glacial 99.5%, 17.4 N R30 Dilute 200 ml glacial acetic acid to 1000 ml with demineralized water (DMHaO). R4 Nitric acid (HN03) 68-70%, 15.5 N R4D Dilute 20 ml cone. HN0s to 1000 ml with DMH20 R5 Ethylenediaminetetraacetic acid (EDTA), fw 292.24 Stock Mehlich 3: Put about 120 ml DMHaO in a 200 ml volumetric flask. Add 27.78 g R2 and mix, then add 14.61 g EDTA, dissolve and make to 200 ml. Mix well and immediately transfer to a plastic bottle. It is necessary to dissolve the R2 first and then add the EDTA to get the EDTA into solution. Extractant: Use a plastic carboy with outlet, calibrated to 4 L, and add about 3 L of DMHaO. Add 80.0 g Rl and dissolve. Add 16 ml Stock Mechlich 3 and mix. Measure with a plastic graduated cylinder. Add 230 ml R3D. Add 164 ml R4D. Make to 4 L with DMHaO and mix thoroughly. pH should be 2.5+0.1. 212 APPENDIX I. FOLIAR NUTRIENT GUIDELINES FOR THE INTERPRETATION OF NUTRITIONAL STATUS FOR HEMLOCK (FROM BALLARD AND CARTER (1986). Interpretation N% P% K% 0 0 0 Very severely deficient Severely deficient Slight moderate deficiency Adequate Severely deficient Moderate-severely deficient Possible slight-moderate de ficiency Little, if any deficiency No deficiency 1.05 0.08 0.35 1.3 0.1 0.45 1.45 0.15 0.75 Ca% Mg% 0 0 0.1 0.06 0.15 0.08 0.2 0.1 0.25 0.12 213 Appendix I (continued) ug S"1 Mn Severe deficiency Probable deficiency Possible deficiency or near deficiency No deficiency Fe He f i i; i <^ nr,y likely P n •- -a i h ~i t- (1 !• i <'. i (» 11 r. V Low to zero probability of deficiency Active Fe Deficiency likely Deficiency unlikely Zn Probable deficiency Possible deficiency No deficiency Cu Probable deficiency Possible moderate deficiency Possibly somewhat deficient Slight possibility of deficiency 0 4 15 25 0 25 50 0 30 0 10 15 0 1 2 2.6 4 No deficiency Appendix I (continued) B Deficiency likely B possibly deficient; Possible B probably not deficient If N<1.5%, then NID possible If N>1.5%, then HID unlikely No deficiency Appendix I (continued) N/P 0 No P deficiency; NID* unlikely 6.11IN + 0.11 No P deficiency; NID unlikely 12 Possible P deficiency; NID or NAD* possible 16 P deficiency P/Al 0 P/Al suggests P deficiency, unless P> 0.13% 3 No interpretation K/Ca 0 Possible K deficiency 0.5 No interpretation 3.5 High K/Ca suggests desirability of checking for possible Fe deficiency Ca/Mg Ca/Mg is unusually low and may impair growth. If soil parent material is of ultramafic origin, consider possibilities of Mo deficiency and Ni and/or Cr toxicity. No interpretation a. NID = Deficiency inducible by N fertilization; NAD = deficiency may be aggravated by N fertilization. 0 0.8 b. N = N percent, dry mass basis. 216 Appendix I (concluded) S If sulfate-S is not evaluated: data suggests actual or inducible S deficiency. If sulfate-S < 0.01%, possible S deficiency and possible NAD1. If sulfate-S >0.01%, no S deficiency but NID1 possible. Possible S deficiency and If sulfate-S < 0.01%, NID- possible. If H II I. f H I*.«—S > 0.01%, NID unlikely. S deficiency and NID are both unlikely. No S deficiency; NID unlikely. N/S (Not interpreted where total S exceeds 0.14%) No S deficiency; NID unlikely. No S deficiency; NID possible. Possible S deficiency. S deficiency. 0.00 % 0.12 % 0.14 % 0.16 % 0 4.2N + 4.94* 13.6 14.6 Sulfate-S (The following applies only where total S has not been evaluated) 0.000 % Actual or inducible S deficiency; NAD or NID likely. 0.008 % No S deficiency; but NID possible. 0.020 % No S deficiency; but NID unlikely. 0.040 % Very high; possibly N deficient. a. NID = Deficiency inducible by N fertilization; NAD = deficiency may be aggravated by N fertilization. b. N = N percent, dry mass basis. 217 APPENDIX J. CONCENTRATIONS OF ZINC HEMLOCK FOLIAGE. AND MANGANESE IN THE CELLULAR FRACTIONS OF MEANS SAMPLE ZNA MNA ZNB MNB ZNC •ug s~x-1 5.3 603 5.3 447 7.7 2 6.3 1460 7.3 1277 8.0 3 41.0 528 34.0 430 45.3 4 4.0 677 4.3 657 6.4 5 5.3 114 7.3 95 7.3 6 9.3 160 9 . 7 153 9.9 7 8.3 15 8.0 15 10.6 STANDARD DEVIATION 1 1.5 62 2 1.2 32 3 14.8 82 4 0.0 49 5 1.5 3 6 1.2 31 7 1.2 3 1.5 36 1.2 0.6 56 2.0 11.3 75 15.9 0.6 125 0.7 2.1 10 0.6 0.6 18 0.8 2.0 4 1.6 MINIMUM 1 4.0 545 2 5.0 1437 3 24.0 452 4 4.0 622 5 4.0 111 6 8.0 127 7 7.0 13 4.0 407 7.0 7.0 1227 6.0 21.0 387 27.0 4.0 537 5.7 5.0 83 7.0 9.0 137 9.0 6.0 12 8.8 MAXIMUM 1 7.0 668 2 7.0 1497 3 51.0 615 4 4.0 717 5 7.0 117 6 10.0 187 7 9.0 18 7.0 477 9.0 8.0 1337 10.0 41.0 517 55.0 5.0 787 7.0 9.0 102 8.0 10.0 172 10.4 10.0 19 11.7 Appendix J (concluded) SAMPLE MNC ZND MND ZNE MNE 1 740 46.4 661 6.1 1300 2 1717 74.4 1692 7.4 2513 3 602 131.3 668 68.7 4 844 56.4 824 5 .4 1280 5 130 110.9 242 8.1 205 6 198 48.5 232 7.9 315 7 19 • 42.6 39 1.0 9 MNC ZND MND ZNE MNE 1 91 35 .0 123 0.5 96 2 17 35.7 141 0.4 153 3 90 26.6 113 20.5 4 64 25 .3 126 0.3 46 5 6 20.9 35 0.1 15 6 12 21.2 58 0.4 28 7 10 44.2 34 0.1 1 MNC ZND MND ZNE MNE 1 657 16.9 525 5.8 1230 2 1707 50.6 1532 7.1 2380 3 527 108.4 580 45 .0 4 776 31.2 687 5.0 1240 5 127 87.7 217 8.0 190 6 185 26.1 183 7.6 285 7 8.8 0.0 0 0.9 9 MNC ZND MND ZNE MNE 1 837 85 .1 766 6.7 1410 2 1737 115 .4 1800 7.9 2680 3 702 160.5 795 81.5 4 902 81.8 936 5.6 1330 5 137 128.2 282 8.3 220 6 207 68.2 295 8.4 340 7 28.1 88.2 59 1.0 10 Sample 1. Hemlock, site 5, control, 1986. 2. Hemlock, site 5, treatment 6, 1986. 3. Hemlock, site 5, treatment 15, 1986. 4. Hemlock, site 5, 1987. 5. Douglas-fir, site 5, 1987. 6. Amabilis fir, site 5, 1987. 7. Yellow cedar, site 5, 1987. 219 APPENDIX K. SCATTER PLOTS OF HEIGHT INCREMENT VERSUS FOLIAR MASS PER SHOOT. 80 -| U CD 00 CJ) 60 H c <D 40H CD c 20 JZ .CP y = 291.Ox - 440.Ox R = 0.38 Q I i i i i i i i i i I i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | 0.0 0.1 0.2 0.3 0.4 0.5 Foliar Mass Per Shoot 1986 (g) Appendix K.1. Scatter plot of the 1986 foliar mass per shoot the for 1986 site height increment versus 2 . 220 80q E : o DO 0.2 0.4 0.6 0.8 Foliar Mass Per Shoot 1986 (g) Appendix K.2. Scatter plot of the 1986 height increment versus the 1986 foliar mass per shoot for site 3. 221 100n y = 160.Ox - 71.Ox 0 00.40.81.2 1.6 Foliar Mass Per Shoot 1986 (g) Appendix K.3. Scatter plot of the 1986 foliar mass per shoot the for 1986 site height increment versus 4. 222 160.0 n E : o Foliar Mass Per Shoot 1987 (g) Appendix K.4. Scatter plot of the 1987 height increment versus the 1987 foliar mass per shoot for site 4. 223 80.0 -| Appendix K.5. Scatter plot of the 1986 height increment versus the 1986 foliar mass per shoot for site 5. 224 120.0 -i '100.0 -3 oo 80.0 H c 0 60.0 CD ^ 40.0 i ^ 20.0 ^ CD 0.0 y = 206.Ox - 124.Ox R = 0.49 0.0 i11u iii!ii111111iIIIi i i i n i 11 11 i 11 i i i i i M 11 i u 111 11 i i i i i nr 0.2 0.4 0.6 0.8 1.0 1.2 Foliar Mass Per Shoot 1987 (g) Appendix K.6. Scatter plot of the 1987 height increment versus the 1987 foliar mass per shoot for site 5. APPENDIX L. FOLIAR NUTRIENT DATA. MEANS l-85,--85a-853 TRT ZN MN --ug g-1  1 10.9 1284 2 14.2 1131 3 15 .1 3008 4 58 .1 1133 5 149 .7 1180 6 9.4 1354 7 10.5 1175 8 7.9 1129 9 9.6 1008 10 9.2 1082 11 7.2 1022 12 7.6 1002 STANDARD DEVIATION !T ZN MN 1 5.6 330 2 9.5 367 3 5.8 1298 4 15 .1 292 5 46 .8 327 6 2.5 416 7 3.4 325 8 2.1 352 9 4.0 388 10 2.4 426 11 1 . 7 321 12 1.9 306 P FE CU B Cg g pg g 0.199 37.8 2.1 13.6 0 .225 40.1 2.4 13 .3 0.181 41.5 2.9 14.4 0.177 40 .2 2.5 14.5 0.174 41.6 2.1 13 .5 0 .192 40 .9 2.4 15 .4 0 .176 43.3 2.4 13.3 0.185 42.2 2.2 13.3 0.191 41.4 2.2 16.5 0.187 39.5 2.3 13.6 0.168 40.3 2.2 13.8 0.185 38 .9 2.4 14.2 P FE CU B 0 .043 5.4 0.6 3.6 0 .057 5.3 0.6 4.4 0 .060 5.4 0 . 7 5.3 0.020 3.7 0.4 3.5 0 .041 4.9 0.3 2.9 0.032 6.2 0.6 4.4 0 .025 3.5 0 . 7 2.6 0 .034 11.3 0.5 4.3 0.038 5.9 0.6 9.0 0 .019 4.2 0.6 3.2 0.024 4.1 0.8 4.8 0 .034 5.2 0.7 6.3 1. Year of fertilizer treatment. 2. Year of foliage collection. 3. Year in which foliage was formed. Appendix L (continued). NUMBER OF CASES TRT ZN MN 1 10 10 2 9 9 3 8 8 4 8 8 5 7 7 6 10 10 7 8 8 8 9 9 9 10 10 10 9 9 11 10 10 12 10 10 MEAN TRT SOS TOTS FLWTBR Mg g _1 eg g~l g 1 0.236 2 0 .221 3 0.360 4 0 .180 5 0.241 6 0 .175 7 342 0.146 0 .202 8 509 0.160 0 .208 9 671 0.165 0 .239 10 851 0.190 0.219 11 460 0.131 0.188 12 401 0.124 0 .283 P FE CU 10 10 10 9 9 9 8 8 8 8 87 7 7 10 10 10 8 8 8 9 9 9 10 10 10 9 9 9 10 10 10 10 10 10 BI1 HTI1 HTI2 0.80 0.73 0.91 0.64 0.60 1.18 0.84 0.78 2.02 0.59 0.65 1.15 0.80 0.51 2.54 0.63 0.69 1.26 0.77 0.69 0.97 0.76 0.73 0.83 0.74 0.72 1.08 0.75 0.61 0.89 0.71 0.56 0.77 0.81 0.62 0.79 Appendix L (continued). STANDARD DEVIATION TRT SOS TOTS FLWTBR 1 0.121 2 0.099 3 0 .246 4 0.042 5 0 .154 6 0.059 7 204 0.024 0.062 8 182 0.012 0.076 9 264 0.019 0 .133 10 112 0.044 0.163 11 130 0.007 0 .050 12 81 0 .013 0.127 NUMBER OF CASES TRT SOS TOTS FLWTBR 1 0 0 10 2 0 0 9 3 0 0 8 4 0 0 8 5 0 0 7 6 0 0 10 7 8 8 8 8 8 8 9 9 10 10 10 10 9 9 9 11 10 10 10 12 - 10 10 10 BI1 HTI1 HTI2 0.29 0.31 0.38 0.15 ; 0.23 0.47 0.32 0.35 1.32 0.14 0.23 0.64 0.28 0.19 1.14 0.17 0.21 0.41 0.17 0.20 0.73 0.16 0.27 0.30 0.22 0.57 0.56 0.25 0.24 0.35 0.18 0.21 0.18 0.17 0.22 0.50 BI1 HTI1 HTI2 10 7 7 9 8 8 8 88 7 7 7 710 9 9 8 8 8 9 9 9 10 10 10 9 8 8 10 9 9 10 10 10 228 Appendix L (continued). 1-85-86-86 HE AN TRT ZN HN P FE CU PS g-X eg S~l pg g"1  1 13.3 1083 0.189 61.2 3.0 2 17.2 915 0.228 67.8 3.6 3 11.0 2971 0.169 62 .9 4.3 4 13.4 1247 0.203 75 .5 3.6 5 15 .2 1142 0.192 70.6 3.3 6 10 .6 1177 0 .200 71.0 3.7 7 11.5 1231 0.184 59.4 2.7 8 11.1 1160 0.184 70.2 2.3 9 9.9 856 0.184 53.6 2.2 10 11.4 914 0.193 58.7 2.4 11 8.8 992 0.197 70.8 3.1 12 9.7 1139 0.207 77.8 3.2 STANDARD DEVIATION TRT ZN HN P FE CU 1 3.9 446 0.041 20 . 7 1.0 2 10.3 285 0.041 33.8 0.7 3 4.6 1278 0 .039 18.4 2 .1 4 2.2 656 0.029 14.0 0.7 5 5.2 382 0.071 29.8 0.8 6 1.8 488 0 .028 32 .5 0.9 7 3.5 352 0 .024 15 .8 0.6 8 3.5 477 0.050 28.1 0.7 9 2.4 407 0 .035 11.4 0 . 7 10 1.5 378 0.034 17.6 1.2 11 2.3 200 0 .040 19 .4 0.6 12 3.2 395 0.045 16 .2 0.9 229 Appendix L (continued). NUMBER OF CASES TRT ZN MN P FE CU 1 7 7 7 7 7 2 9 9 9 9 9 3 8 8 8 8 8 4 7 7 7 7 7 5 9 9 9 9 9 6 8 8 8 8 8 7 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 10 7 7 7 7 7 11 9 9 9 8 8 12 9 9 9 9 9 MEAN TRT B TS FLWTBR BI2 ug g-1 eg g-1- g 1 21.8 0.248 1.26 2 23.8 0.221 1.59 3 19.6 0.348 1.54 4 24.5 0.264 1.22 5 22.9 0.206 1.61 6 22.5 0.193 1.06 7 20.8 0.151 0.202 1.10 8 22.5 0.153 0.140 0.87 9 22.8 0.156 0.213 0.99 10 22.3 0.157 0.343 1.36 11 23.7 0.143 0.159 0.92 12 23.2 0.150 0.195 0.84 Appendix L (continued). STANDARD DEVIATION TRT B TS FLWTBR 1 6.2 0.206 2 4.1 0.120 3 6.1 0.192 4 6.4 3 5 3.6 0.149 6 6.5 1 7 4.0 0.020 0.117 8 7.2 0.021 0.056 9 10.4 0.018 0.131 10 4.0 0.020 0.2311 6.6 0.015 0.061 12 8.3 0.031 0.13NUMBER OF CASES TRT B TS FLWTBR 1 7 0 24 2 9 0 7 3 8 0 24 4 7 0 1 5 9 0 27 6 8 0 4 7 8 8 28 9 9 18 9 9 9 27 10 7 7 18 11 8 7 27 12 9 8 30 BI2 1.02 1.92 0.69 0.51 0.71 0.32 0.52 0.23 0 .33 0 .88 0 .24 0.31 BI2 24 27 24 21 27 24 24 18 27 18 27 30 231 Appendix L (continued). 2-85-85-85 MEAN TRT ZN MN P FE CU Mg g-1 eg g-1- jig g-t  1 6.9 903 0.181 35 .7 2.8 2 7.9 1403 0 .126 37.7 2.9 3 7.2 3142 0.111 36.5 3.1 4 58.8 164.8 0.188 40.0 3.2 5 75 .0 1230 0.124 35 .7 3.6 6 9.0 1380 0.163 44.6 3.3 7 6.7 1167 0.141 35.7 3.0 8 5.8 1140 0.163 37.1 2.8 9 8.3 1343 0.136 35 .7 2.7 10 8.5 1268 0.167 40.8 3.0 11 7.0 1030 0.136 40.5 3.1 12 6.9 1449 0 . 126 38 .3 3.1 STANDARD DEVIATION TRT ZN MN 1 2.8 393 2 1.6 489 3 3.3 1470 4 21.8 548 5 99.0„. 99 6 3.0 328 7 2.3 794 8 2.8 667 9 6.2 401 10 2.3 572 11 2.4 172 12 2.5 655 P FE CU 0.050 4.6 0.5 0.044 6.5 0.8 0.021 4.6 0.4 0.056 3.0 0.4 0.042 0.053 7.4 0.9 0.037 7.1 0.4 0.046 5.4 0.8 0.036 12.9 0.5 0.060 6.1 0.3 0.027 2.1 1.1 0.040 4.0 0.8 232 Appendix L (continued). NUMBER OF CASES TRT ZN MN P FE CU 1 7 7 7 7 7 2 8 8 8 7 7 3 9 9 9 9 9 4 5 5 5 5 5 5 2 2 2 1 1 6 6 6 6 4 4 7 3 3 3 3 3 8 5 5 5 5 5 9 8 8 8 7 7 10 8 8 8 7 7 11 4 4 4 3 3 12 9 9 9 7 7 [EAN B TOTS SOS FLWTBR BI1 HTI2 HTI1 Ug 6"1 eg g~l ug g"1 S 13.9 0.143 0.76 1.29 1.05 17.5 0.129 0 . 76 1 .92 0 . 77 17.3 0.145 0.82 1.82 0 .90 16 .6 0 .153 0.86 1.57 0.72 18.2 0.077 0.59 1.85 0.55 17.3 0.136 0.79 1.32 0.83 15 .7 0.119 407 0.107 0.74 1.42 0.75 18.6 0 .151 635 0.118 0 . 75 1.57 0.91 19.6 0.141 585 0.144 0.87 1.50 0.85 17.1 0.160 837 0.168 0.72 1.35 0.77 17.1 0.624 505 0.136 0.73 1 .38 0.63 18 .9 0.273 621 0.143 0 . 71 1 .20 0.94 Appendix L (continued). STANDARD DEVIATION B TOTS SOS FLWTBR BI1 HTI2 HTI1 3 . 7 0 .048 0.16 0.58 0 .35 5.4 0.040 0.14 0.96 0.35 4.0 0 .056 0.15 0.51 0 .12 3.2 0.034 0.22 0.85 0.32 3.9 0.041 1.62 0.16 8.7 0.048 0.18 0.66 0.33 2.4 0.027 51 0.019 0.01 0.41 0.28 3.4 0.023 305 0.042 0.21 0.20 0.54 2.7 0 .012 149 0 .037 0.28 0 .39 0.18 5.7 0.019 109 0.059 0.11 0.68 0.39 8.1 0 .889 370 0.054 0.08 0 . 73 0.14 4.2 0.395 135 0.049 0.15 0.45 0.34 IHBER OF CASES B TOTS SOS FLWTBR BI1 HTI2 HTI1 7 0 0 7 7 6 6 7 0 0 8 8 7 7 9 0 0 9 9 7 7 5 0 0 5 5 7 7 2 0 0 2 1 5 6 6 0 0 6 6 4 4 3 2 2 3 3 3 3 5 5 5 5 5 5 5 7 8 8 8 8 6 6 8 8 7 8 8 4 5 4 3 3 4 4 4 4 8 8 8 9 9_ 7 7 234 Appendix L (continued). 2-85-86-86 MEAN TRT ZN MN P FE CU Ug g-X eg S~x pg g-* 1 13.3 1323.8 0 .145 53.7 2.4 2 13.6 1399.3 0.148 42.9 2.3 3 12.4 3875.1 0.128 39.3 2.5 4 11.5 1526.7 0.153 55 .6 3.1 5 19 .6 1140.8 0.130 52.8 2.7 6 12.7 1327.8 0.118 51.4 2.8 7 8.8 1727.5 0.140 31.4 2.6 8 11.4 1180 .8 0 .152 35.1 2.7 9 11.4 1401.4 0.136 33.8 2.6 10 13.0 2088 .0 0 .132 32.5 2.9 11 10.1 1249.3 0.125 57.1 2.9 12 11.1 1931.8 0.126 41.4 2.8 1ARD DEVIATION TRT ZN MN P FE CU 1 3.8 289 .3 0 .064 18.0 0.4 2 2.7 433 .9 0.042 14.4 0.9 3 5.6 1428.5 0 .042 13.2 0.6 4 5.6 1031.0 0.035 10.3 1.0 5 8.8 433.6 0.055 9.6 0 . 7 6 3.7 370.9 0 .024 23.3 0.5 7 3.4 1025 .1 0.028 7.7 0.9 8 2.7 601.0 0.042 5.4 0.6 9 6.8 359.5 0.033 8.0 0 . 7 10 5.5 985 .4 0.027 7.1 0.7 11 4.2 357.4 0.029 14.3 0.9 12 4.8 589.0 0.029 7.8 0.4 235 Appendix L (continued). NUMBER OF CASES TRT ZN 1 6 23 9 4 7 5 5 67 4 8 6 9 7 10 5 11 4 12 8 MN 6 6 9 7 5 5 4 6 7 5 4 8 P 6 6 9 7 5 5 4 6 7 5 4 8 FE 6 6 9 7 5 5 4 6 7 5 4 8 CU 6 6 9 7 5 5 4 6 7 5 4 8 MEAN TRT B TOTS FLWTBR BI2 Ug g~l eg g"1 g 1 28.8 0.140 1.31 2 25 .8 0.196 1.58 3 33.0 0.181 1.42 4 28 . 7 0.158 1.58 5 26.5 0.131 1.34 6 30.8 0.190 1 .06 7 37.1 0.147 0.123 1 .23 8 22 .9 0 .156 0.163 1 .08 9 30.0 0.153 0.140 1 .28 10 31.3 0.162 0.156 1.14 11 31.0 0.140 0.163 1.47 12 30.0 0.148 0.186 1 .02 236 Appendix L (continued). STANDARD DEVIATION TRT B TOTS FLWTBR BI2 I 7.5 0 .047 0.48 2 7.6 0.119 0.53 3 9.4 0 .096 0.55 4 5.0 0.068 0.68 5 6 . 7 0.092 0.51 6 4.1 0.104 0.25 7 13 .1 0.012 0.039 0 .45 8 4.9 0.019 0.113 0.51 9 3.8 0 .008 0.057 0.40 10 6.1 0.019 0.147 0.53 11 10.2 0 .008 0.074 0.76 12 5.0 0.012 0.076 0 .33 NUMBER OF CASES TRT B TOTS FLWTBR BI2 16 0 18 18 2 6 0 8 18 3 9 0 27 27 4 7 0 18 18 5 5 0 5 15 6  0 14 14 7 4 3 2 12 8 6 5 18 15 9 7 6 24 24 10 5 5 15 15 11 4 4 2 12 12 8 6 24 24 Appendix L (continued). 3-85-86-86 MEAN TRT ZN MN P B TOTS pg g-i eg g"1 pg g-x eg g" 1 11.1 1502 0.174 16.2 2 17.7 1638 0 .193 25 . 7 3 15 .5 2427 0 .187 22.5 4 21.3 2019 0 .220 27.8 5 19.1 1793 0.178 21.7 6 15 .4 1899 0.173 27.8 7 14.4 2197 0.230 28 .1 0.158 8 17.4 2268 0.170 28.7 0.140 9 13.1 1245 0.185 28.2 0.145 10 12 .8 1212 0 .173 26.6 0.140 11 17.9 1853 0.180 22.7 0.143 12 11 . 7 1505 0 . 188 23.6 0 .140 STANDARD DEVIATION TRT ZN MN P B TOTS 1 1.9 455 0.026 3.2 2 10.0 648 0.028 5.4 3 2 .4 441 0.010 4.6 4 2.4 994 0.036 8.7 5 8.3 775 0.049 8.7 6 6.7 839 0.039 10.2 7 3.7 331 0.047 5 . 7 0.023 8 3.4 1037 0.038 12.2 0.029 9 3.5 280 0.021 4.5 0 .021 10 4.3 795 0.022 9.1 0.024 11 5.5 547 0 .026 4.8 0.047 12 1.7 287 0.033 9.2 0.023 Appendix L (continued) NUMBER OF CASES TRT ZN 1 7 23 7 4 3 5 8 67 8 89 4 1011 8 12 6 MN 7 7 7 3 8 8 8 8 4 4 8 6 P 7 7 7 3 8 8 8 8 4 4 8 6 B 7 6 7 3 8 8 8 8 4 4 8 6 TOTS 0 0 0 0 0 0 8 8 4 4 8 6 MEAN TRT FLWTBR 8 BI2 BI1 HTI2 HTI1 1 2 3 4 5 6 7 8 9 10 11 12 0 0 0 0 0 0 0 0 .241 ,303 ,308 255 ,193 214 ,171 172 0.252 0.303 0.315 0.262 0.90 0.87 0.99 1.02 1.02 1 .00 0.86 0 . 79 1.02 0.92 1.05 0.95 0 .94 1.06 1.09 1 .18 0.78 0.93 0.86 0.82 0.93 1.09 0.90 0.88 1 1 1 0 1 1 0 1 0.97 1.17 1.20 1.28 04 16 05 99 10 07 79 09 1.01 1.00 1.07 1.15 0.92 1.07 1.00 0.84 0 .80 1 .18 0.94 0.83 Appendix L (continued). STANDARD DEVIATION TRT FLWTBR BI2 1 0 .106 0 .28 2 0.248 0.23 3 0.175 0.23 4 0.125 0.49 5 0.081 0.32 6 0.087 0.50 7 0 .089 0.37 8 0.105 0.30 9 0 .072 0.57 10 0.360 0.26 11 0.210 0 .35 12 0.085 0.26 NUMBER OF CASES TRT FLWTBR Bl: 1 21 21 2 21 21 3 21 21 4 12 12 5 23 23 6 22 22 7 27 27 8 26 26 9 12 12 10 15 15 11 27 27 12 24 24 BI1 HTI2 HTI1 0 .35 0.58 0.28 0.33 0.48 0.29 0.40 0.27 0 .36 0.20 0.21 0.41 0.38 0.37 0.44 0.34 0.54 0.22 0.27 0.22 0.45 0.23 0.30 0.45 0.24 0.07 0.23 0.37 0.47 0.37 0.42 0.46 0.36 0.26 0.53 0.38 BI1 HTI2 HTI 21 8 8 21 6 6 21 8 8 12 3 3 23 8 8 22 6 6 24 9 9 26 9 9 12 4 4 15 5 5 24 9 9 24 8 8 Appendix L (continued). 4-86-86-86 MEAN TRT ZN MN P6 g~l ~ 1 10.9 1469 2 8.7 1112 3 11.8 1098 4 9.2 2026 5 11.4 2802 6 9.1 3206 7 8.8 1012 8 9.6 1360 9 8.1 1168 10 11.3 1639 11 10.7 1647 12 11.8 1175 13 18.3 1448 14 26 .2 1384 15 48.6 1536 16 9.1 1208 17 7.7 1169 18 8.2 1464 19 9.4 1389 20 6.4 986 21 10.4 1411 22 7.8 1433 23 17.2 1343 24 9.2 1432 25 8.7 1428 N p -1 MG cg g 1.07 0 .170 0 .136 0.99 0 .175 0 .125 0.97 0 .153 0 .118 1.18 0 . 120 0 . 140 1.30 0 .125 0 . 140 1.33 0 . 138 0 .118 1.05 0 .160 0 .135 1 .04 0 .178 0 .140 0.94 0 .150 0 .128 1.09 0 .184 0 .136 0.98 0 .178 0 .158 1 .16 0 .134 0 .126 1.10 0 .163 0 .130 1.04 0 . 155 0 . 123 1.06 0 .178 0 .148 0 .99 0 . 160 0 . 126 1.01 0 .148 0 .120 0.97 0 .155 0 . 143 1.13 0 .148 0 .123 0.91 0 .118 0 .110 1.09 0 .162 0 .124 0.97 0 .135 0 .138 1.67 0 .160 0 .118 1.12 0 .136 0 . 130 1.00 0 .197 0 .140 Appendix L (continued) STANDARD DEVIATION TRT ZN MN PS s~r  1 4.5 445 2 2.3 378 3 4.6 264 4 1.7 727 5 4.5 710 6 1.5 1341 7 2.9 257 8 2.9 432 9 2.6 456 10 4.3 597 11 6.0 604 12 3.6 214 13 6.9 247 14 10.4 283 15 25 .1 724 16 2.0 415 17 3.3 287 18 3.3 550 19 3.1 112 20 2.1 239 21 4.3 613 22 2 . 1 310 23 6.2 400 24 2.6 410 25 1.9 360 N P MG -eg g 0 .28 0 .010 0 .025 0 .18 0 .053 0 .024 0 .12 0 .051 0 .022 0 .07 0 .023 0 .016 0 .13 0 .035 0 .014 0 .18 0 .015 0 .029 0 .35 0 .042 0 .029 0 .29 0 .036 0 .008 0 .13 0 .042 0 .011 0 .23 0 .040 0 .034 0 .25 0 .050 0 .017 0 .25 0 .038 0 .034 0 .20 0 .057 0 .029 0 .09 0 .047 0 .015 0 .32 0 .073 0 .051 0 .20 0 .034 0 .023 0 .24 0 .031 0 .021 0 .35 0 .013 0 .010 0 .17 0 .039 0 .017 0 .17 0 .025 0 .014 0 .23 0 .040 0 .015 0 .13 0 .006 0 .022 0 .26 0 .014 0 .022 0 .19 0 .030 0 .025 0 .06 0 .071 0 .062 242 Appendix L (continued) NUMBER OF CASES TRT ZN MN N P MG 1 5 5 5 5 5 2 4 4 4 4 4 3 4 4 4 4 4 4 5 5 5 5 5 5 4 4 4 4 4 6 4 4 4 4 4 7 4 4 4 4 4 8 4 4 4 4 4 9 5 5 5 5 5 10 5 5 5 5 5 11 4 4 4 4 4 12 5 5 5 5 5 13 4 4 4 4 4 14 4 4 4 4 4 15 4 4 4 4 4 16 5 5 5 5 5 17 5 5 5 5 5 18 4 4 4 4 4 19 4 4 4 4 4 20 4 4 4 4 4 21 5 5 5 5 5 22 4 4 4 4 4 23 4 4 4 4 4 24 5 5 5 5 5 25 3 3 3 3 3 Appendix L (continued) MEAN FE CU B AFE FLWTBR BI2 — l. 8 --ug g 30.7 3.1 26.2 37.0 0.193 0.91 24.1 3.0 23 .1 32.3 0 .253 0.91 38.4 3.1 24.1 28.8 0.225 1 .18 25 .0 3.6 22.5 31.8 0.270 1.03 24.1 3.7 23.4 31.9 0.352 1.15 33.9 3 . 7 23.0 36.3 0.363 1.18 30.9 2.5 36.7 30.8 0.229 0.92 27.4 2.7 36 .9 30.8 0 .308 0 .92 35.7 2.3 38.8 31.8 0.191 0.85 32 .1 2.9 42 .2 32.5 0.271 0 .83 32 .1 2.5 89.5 32 .6 0.197 0.85 33 .6 2.6 79.0 32 .0 0.287 0.90 29.5 3.7 24.3 34.9 0.263 0.98 25 .9 3.8 24.0 35 .0 0.299 0.84 31.2 3.1 25.1 33.0 0.171 0.79 25 .0 3.9 25.7 30 .2 0 .286 0.83 32 .1 4.0 22 .8 35.2 0.211 0.82 34.8 2.6 23.8 32 .0 0 .216 0 .95 35 .7 2.7 21.8 30.8 0.267 0.80 28.6 2.3 22 . 7 27.5 0.136 1.04 34.3 2.9 26.8 30 .6 0.317 0.94 33.0 2.9 22.9 30.8 0.243 0.94 38.4 3.5 33.2 37.2 0.804 1.23 32.1 2.6 26 .0 30.2 0.198 0.78 25.0 , 3.2 24.5 32 .0 0.200 0.76 Appendix L (continued) STANDARD DEVIATION FE CU B AFE FLWTBR BI2 8 pg g 12 .8 1.0 4.1 9.3 0.100 0.39 8.9 0.6 7.0 3.9 0.170 0.21 29.9 0.4 6.1 5.8 0.083 1.13 5.0 0.4 6.3 3.8 0.096 0.26 8.4 0.9 2.8 5.5 0.388 0.38 8.5 0.4 2.5 3.3 0 .284 0.37 4.1 0.6 6.7 4.2 0.112 0.27 2 .1 0.7 15 .2 1. 7 0.2 76 0.31 11.6 0.5 7.5 2.0 0.080 0.34 5.6 0.3 11.0 4.4 0.178 0.27 12.4 0.8 30.7 6.0 0.058 0.28 6.0 0.6 21.6 4.1 0.239 0.32 10.3 0.7 2.4 7.1 0.156 0.47 5.4 0.6 4.9 5.7 0.082 0.39 4.5 0.8 5.1 5.4 0.136 0.35 5.6 0.9 3.7 4.0 0.231 0.29 9.2 1.2 9.6 8.4 0.073 0.24 3.4 0.5 6.8 1.6 0 .231 0.46 9.2 0.5 2.5 5.5 0.071 0.17 5.8 0.8 3.2 5.8 0.063 0.37 7.4 0.5 2.7 6.2 0.239 0.29 6.1 0.6 3.7 3.5 0 .062 0.46 1.8 0.6 4.1 2.4 0.646 0.48 5.6 0.7' 2.9 1.8 0 .120 0 .28 1.8 0.127 0.22 Appendix L (continued) NUMBER OF CASES FE CU B AFE FLWTBR BI2 5 5 5 5 5 30 4 4 4 4 4 27 4 4 4 4 4 27 5 5 5 5 5 30 4 4 4 4 4 24 4 4 4 4 4 26 3 3 3 3 4 27 3 3 3 3 4 21 5 5 5 5 5 26 5 5 5 5 5 30 4 4 4 4 4 30 5 5 5 5 5 24 4 4 4 4 4 30 4 4 4 4 4 21 4 4 4 4 4 25 5 5 5 5 5 27 4 4 4 4 5 28 4 4 4 4 4 27 4 4 4 4 4 30 4 4 4 4 4 27 5 5 5 5 5 30 4 4 4 4 4 27 4 4 4 4 4 30 5 5 5 5 5 30 1 1 3 1 3 24 Appendix L (continued) 4-86-87-87 MEAN TRT ZN MN Pg 8"1  1 11.7 1719 2 8.5 1302 3 11.2 1465 4 10.1 2980 5 9.2 3460 6 9.7 3737 7 8.8 1525 8 10.6 1804 9 6.4 1452 10 13.8 1598 11 11.6 1743 12 10.2 1593 13 10.2 1412 14 8.3 1407 15 9.4 1481 16 9.0 1409 17 9.4 1644 18 8.5 1577 19 9.0 1586 20 9.7 1635 21 8.5 1512 22 8.5 1772 23 11.1 1259 24 9.7 1587 25 11.7 1545 N P MG eg g 1• 1 .15 0.193 0.140 1 .03 0.198 0.149 1 .01 0.184 0.141 1 . 12 0.157 0.150 1 .12 0.163 0.130 I .19 0.167 0.116 1 .10 0.184 0.147 1 .19 0.207 0 .143 0 .96 0.169 0.149 1 .20 0.197 0 .145 1 .16 0.198 0.152 1 .18 0.159 0.154 1 .12 0.176 0.148 0 .96 0.169 0.146 1 .04 0.160 0.167 I .16 0.164 0.154 1 .28 0.166 0.135 1 .06 0.165 0.143 1 .14 0.172 0.151 1 .25 0 .168 0.137 0 .99 0.161 0.137 1 .11 0.178 0 .170 1 .22 0.208 0.143 1 .16 0.157 0.163 1 .17 0.183 0.159 Appendix L (continued) STANDARD DEVIATION TRT ZN HN Ug g-x 1 5.8 511 2 1.8 432 3 2.7 447 4 4.2 918 5 2.5 780 6 3.8 1237 7 3.2 747 8 4.2 689 9 2.3 428 10 8.9 412 11 6.0 471 12 3.5 365 13 2.6 454 14 2.6 263 15 3.0 352 16 3.1 542 17 3.5 544 18 3.0 332 19 3.1 270 20 ' 3.5 375 21 3.9 281 22 2.0 343 23 3.5 305 24 2.7 363 25 4.6 415 N P HG eg g 0 .27 0.040 0 .030 0 .20 0 .045 0 .031 0 .20 0.055 0.033 0 .17 0.025 0 .021 0 .19 0.035 0.022 0 .21 0.034 0.023 0 .31 0.064 0.025 0 .27 0.043 0.015 0 .22 0.034 0.021 0 .25 0.026 0 .029 0 .21 0.042 0.024 0 .29 0 .047 0.012 0 .12 0.026 0.027 0 .20 0 .038 0.027 0 .27 0.051 0.031 0 .12 0.029 0 .033 0 .25 0.029 0.028 0 .27 0 .024 0 .021 0 .22 0.049 0.034 0 .22 0.048 0.031 0 .22 0.039 0.022 0 .20 0 .035 0.026 0 .15 0.024 0.029 0 .27 0 .031 0 .022 0 .21 0.041 0.040 248 Appendix L (continued) NUMBER OF CASES TRT ZN MN N P MG 1 9 9 9 9 9 2 9 9 9 9 9 3 9 9 9 9 9 4 10 10 9 10 10 5 9 9 9 9 9 6 9 9 9 9 9 7 10 10 10 10 10 8 7 7 7 7 7 9 9 9 9 9 9 10 10 10 10 10 10 11 9 9 9 9 9 12 8 8 8 8 8 13 9 9 9 9 9 14 8 8 8 8 8 15 8 8 8 8 8 16 7 7 7 7 7 17 10 10 10 10 10 18 8 8 8 8 8 19 9 9 9 9 9 20 10 10 10 10 10 21 9 9 9 9 9 22 9 9 9 9 9 23 9 9 9 9 9 24 10 10 10 10 10 25 9 9 9 9 9 Appendix L (continued) MEAN FE CU FLWTBR BI3 HTI3 HTI2 ug g-i 6 50.4 3.4 0.523 1.42 1.86 1.31 46.4 3.1 0.40 7 I .15 1.59 1 .15 52.0 3.2 0.364 1.14 1.59 1 .40 47.5 3.4 0.449 1.91 3 .14 1.21 50.4 3.3 0.506 1.77 2.65 1.32 55.9 3.6 0.646 1 .33 2.27 1.20 46.4 3.4 0.431 1.43 1.76 1.18 50.5 3.5 0.592 1.80 1.98 1.04 41.7 3.1 0.199 1.24 1.82 0.87 49.3 3.3 0 .665 1 .62 2.57 1.11 48.0 3.8 0.45 7 1.62 2.26 1.24 46.4 3.7 0 .428 1 .35 1.98 1.26 42.8 3.8 0.421 1.86 3.30 0.98 43 .3 3.4 0.371 1.75 2 .39 0.78 37.9 3.6 0.368 1.43 1.85 0.99 47.4 3.8 0.505 1.51 I .66 1 .04 54.3 3.6 0.540 1.66 1.75 1.19 46 .0 3.6 0.327 1.14 2 .35 1.01 41.7 3.9 0.459 1.46 2.11 0.98 43 .6 4.0 0 .515 1.30 1.60 1.25 44.8 3.3 0.410 1.46 2.00 0.83 46.8 3.7 0 .321 2.27 1.95 1.14 51.2 3.8 0.635 1.87 2.60 1.45 47.1 3.6 0.344 1.51 3.17 1.19 48.8 3.6 0.280 1.77 1.93 1.31 Appendix L (continued) STANDARD DEVIATION FE CU FLWTBR BI3 HTI3 HTI2 —PS g-l S 10.9 1.2 0.318 0.33 0.79 0.61 9.1 i.l 0.285 0 .36 0.50 0.60 12.5 0.9 0.195 0.51 1.01 0.89 18 .4 0.7 0 .219 0.65 2.29 0.77 20.2 0.7 0.358 0.59 1.55 0.77 9.8 0.8 0.459 0.50 1.57 0.56 12.0 1.1 0 .295 0.45 0.61 0 .40 10 .6 1.0 0.411 0.51 0 .65 0 .49 8.9 0.7 0.084 0.35 0.84 0.54 10.6 0.7 0.427 0.72 1.99 0.89 19.4 0.8 0.350 0.46 1.34 0.61 8.5 0.9 0.316 0.70 1.23 0.52 16.2 0.9 0.340 1.22 3.02 0.67 13 .3 0.9 0.226 0.70 3 .13 0.59 7.6 0.8 0.293 0.32 1.02 0.50 7.9 0.6 0.288 0.62 1.00 0.27 12.7 1.0 0.266 0.37 0.63 0.48 12.7 1.1 0.174 0 .39 1.66 0.31 8.4 0.7 0.277 0.20 1.25 0.48 6.3 0.9 0 .335 0.34 0.41 0.59 12.1 0.7 0.329 0.40 1.00 0.42 12.3 0.9 0.166 2.37 1.04 0.83 10.6 0.6 0.346 1.25 2.50 0.67 12 .7 0.9 0.233 0 .30 3 .18 0.80 15 .4 0.6 0.145 0.59 0.57 0.62 Appendix L (continued) NUMBER OF CASES FE CU FLWTBR BI3 HTI3 HTI2 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 10 9 10 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 10 10 10 7 7 7 7 7 7 9 9 9 9 9 9 10 10 10 10 10 10 9 9 9 9 9 9 8 8 8 8 8 8 9 9 9 9 9 9 8 8 8 8 7 8 8 8 8 8 8 8 7 7 7 7 7 7 10 10 10 10 10 10 8 8 8 8 8 8 9 9 9 9 9 9 10 10 10 10 10 10 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 10 10 10 9 9 9 9 8 9 Appendix L (continued) 5-86-86-86 MEAN TRT ZN MN ug g"1  1 10.7 1262 2 11.6 1273 3 14.3 1289 4 11.9 1832 5 12.1 2172 6 15 .7 2933 7 10.1 1178 8 12.0 1525 9 8.8 1327 10 8.3 1083 11 10.5 1268 12 10.1 1240 13 79.3 1217 14 100.9 1066 15 132.3 867 16 14.1 1251 17 10.1 1425 18 11.2 1140 19 10.1 1272 20 10 .5 1094 21 11.2 1008 22 10.1 1270 23 15.5 1064 24 8.6 1091 25 11.4 1308 N P MG — -eg g 1 .02 0.155 0 .123 1 .02 0.193 0 .140 1 .12 0.183 0 .137 1 .21 0.153 0 .105 1 .14 0.176 0 .100 I .35 0.164 0 .124 1 .06 0.158 0 .125 1 .10 0.204 0 .134 0 .98 0.167 0 .150 0 .92 0.178 0 .140 1 .05 0.195 0 .140 0 .99 0.213 0 . 143 1 .26 0.188 0 .140 1 .13 0.195 0 .108 1 .04 0.188 0 .138 1 .08 0.182 0 . 106 1 .09 0.207 0 .137 I .21 0.192 0 .118 1 .04 0.182 0 .116 1 .02 0.178 0 .133 1 .16 0.176 0 .124 1 .01 0.150 0 .165 1 .67 0.214 0 .136 1 .02 0.138 0 .134 1 .11 0.182 0 .140 Appendix L (continued) STANDARD DEVIATION TRT ZN HN PS s-1  1 5.8 137 2 4.9 209 3 7.1 548 4 2.3 510 5 5.2 794 6 5.8 1021 7 2.7 416 8 2.9 670 9 1.6 536 10 1.5 363 11 2.7 408 12 1.4 526 13 30.1 235 14 75.8 319 15 30.5 487 16 4.4 509 17 3.7 535 18 4.8 249 19 3.3 751 20 1.9 261 21 3.3 174 22 2.1 249 23 4.2 370 24 1.9 672 25 1.9 531 253 N P NG eg g 0.27 0.042 0.013 0.17 0.017 0 .050 0.26 0.025 0.012 0.08 0.041 0.019 0.24 0.036 0.012 0. 12 0 .025 0.040 0.34 0.032 0.019 0 .14 0.073 0.026 0.05 0.055 0.044 0.12 0.056 0.026 0.19 0.048 0.034 0.22 0.049 0.033 0.16 0.028 0.018 0.27 0.031 0 .022 0.24 0.043 0.019 0.14 0.047 0.011 0.15 0.055 0.023 0.24 0 .054 0.022 0.13 0.015 0.015 0.06 0.043 0.026 0.19 0.017 0.026 0.17 0.024 0.040 0.22 0.066 0.052 0.15 0.030 0.038 0.11 0.052 0.016 254 Appendix L (continued) NUMBER OF CASES TRT ZN MN N P MG 1 4 4 4 4 4 2 4 4 4 4 4 3 3 3 3 3 3 4 4 4 4 4 4 5 5 5 5 5 5 6 5 5 5 5 5 7 4 4 4 4 4 8 5 5 5 5 5 9 3 3 3 3 3 10 4 4 4 4 4 11 4 4 4 4 4 12 4 4 4 4 4 13 4 4 4 4 4 14 4 4 4 4 4 15 5 5 5 5 5 16 5 5 5 5 5 17 3 3 3 3 3 18 5 5 5 5 5 19 5 5 5 5 5 20 4 4 4 4 4 21 5 5 5 5 5 22 4 4 4 4 4 23 5 5 5 5 5 24 5 5 5 5 5 25 5 5 5 5 5 Appendix L (continued) MEAN FE CU B AFE FLWTBE BI2 S PS 6 40.2 2.9 22.3 26.7 0.169 1.01 4i .9 2.9 22 .1 27.8 0.201 1 .03 40.5 3.3 23.1 25 .9 0.171 0.87 45 .5 3.6 20.1 30 .2 0 .302 1.25 40.0 3.1 24.2 29.2 0.250 1.21 40.0 3.6 23 .9 28.2 0.367 1.23 32.1 3.5 35.7 28.5 0.256 1.08 32.1 3.0 38 .5 25.8 0.175 0.99 44.0 2.7 47.1 27.7 0.189 0.86 32.1 3.0 40.9 24.2 0.193 1.09 36.6 2.9 51.9 25.6 0.168 0.98 36 .6 3.1 52.6 27.3 0.250 1.08 46.4 4.9 22.1 30.0 0.184 0.97 36 .6 3.0 22.4 30 .3 0.173 0.87 40.7 2.8 26.8 32.6 0.171 0.83 39.3 3.1 21.1 31.3 0.277 1.10 45 .2 3.8 24.5 33.9 0.160 0.95 36 .4 3.8 23.3 32.1 0.291 0 .98 32.1 3.5 24.9 28.9 0.198 0.93 32.1 2.9 25 .5 29. 7 0 .174 0 .94 32.1 3.4 24.8 .26.4 0 .200 0.98 30 .3 3.2 23.5 29.6 0.181 0.93 46.4 4.0 45 .8 34.8 0.394 1.58 51.4 3.6 23.2 32.7 0 .146 0.88 39.3 3.1 28.4 26.2 0.198 0.96 256 Appendix L (continued) STANDARD DEVIATION FE CU B AFE FLWTBR BI2 Ug g 1- 8 12.2 0.7 1.3 7.4 0.045 0.26 6.1 0.6 7.6 6.2 0.081 0 .38 8.2 0.2 3.5 3.0 0.028 0.32 11.8 0.7 5.2 3.2 0.096 0.35 8.9 0.7 4.8 7.7 0.134 0.52 6.4 0.7 5.3 4.2 0.187 0 .39 13.0 1.6 12.2 3.1 0.107 0.36 7.1 0.6 17.8 6.3 0.078 0.31 7.4 0.5 11.3 4.1 0.021 0.21 14.9 0.4 13.3 9.0 0.075 0.35 3.4 0.3 10.1 5.0 0.037 0.37 5.4 0.3 15 .2 4.4 0.210 0.39 15 .4 1.3 3.0 5.9 0.078 0.22 8.4 l.l 2.3 4.6 0 .052 0.31 15.5 1.3 4.5 10.9 0.093 0.37 11.6 0.4 1.7 5.5 0.149 0.36 10.3 0.5 4.2 9.0 0.045 0.27 8.5 1.0 6.3 6.8 0.072 0.35 12 .6 0.5 5.7 7.6 0.071 0.26 12.7 0.7 9.2 2.4 0.052 0.35 7.1 0.6 5.5 7.3 0.100 0.35 7.4 0.4 1.9 7.0 0 .049 0.23 9.1 0.6 10.9 6.4 0.162 0.67 12.5 0.6 2.4 3.5 0.048 0.21 6.7 0.6 6.0 5.3 0.054 0.27 \ 257 Appendix L (continued) NUMBER OF CASES FE CU B AFE FLWTBR BI2 4 4 4 4 4 24 4 4 4 4 4 27 3 3 3 3 3 27 4 4 4 4 4 27 5 5 5 5 5 25 5 5 5 5 5 27 4 4 4 4 4 24 5 5 5 5 5 30 3 3 3 3 3 23 4 4 4 4 4 24 4 4 4 4 4 27 4 4 4 4 4 24 4 4 4 4 4 24 4 4 4 4 4 24 5 5 5 5 5 26 5 5 5 5 5 30 3 3 3 3 3 21 5 5 5 5 5 27 5 5 5 5 5 21 4 4 4 4 4 29 5 5 5 5 5 30 4 4 4 4 4 24 5 5 5 5 5 24 5 5 5 5 5 30 5 5 5 5 5 27 Appendix L (continued) 5-86-87-87 MEAN TRT ZN MN ug g"1  1 11.9 1190 2 12.6 1296 3 15 .2 1342 4 11.2 2487 5 12.6 2817 6 12.5 3518 7 11.3 1603 8 11.5 1322 9 8.8 1439 10 9.9 1359 11 12.0 1626 12 11.6 1301 13 11.9 1581 14 15 .4 1540 15 16.5 1533 16 11.5 1515 17 9.9 1285 18 10.5 1231 19 12 .5 1463 20 12.4 112 7 21 12.2 1551 22 12 .0 1434 23 14.7 962 24 9.9 1142 25 11.8 1243 258 N P MG -eg g *•• 1 .04 0.163 0 .139 1 .12 0.185 0 .139 1 .04 0.173 0 .148 1 .16 0.157 0 .114 1 .12 0.171 0 .113 1 . 16 0.159 0 .117 1 .20 0.164 0 .133 I .15 0.177 0 .147 1 .03 0.143 0 .140 1 .09 0.159 0 .159 1 .23 0.175 0 .139 1 .12 0.181 0 .160 1 .12 0.165 0 .147 1 .19 0.174 0 .124 1 .22 0.215 0 .154 1 . 10 0.173 0 .133 1 .11 0.162 0 .139 1 .17 0.192 0 .139 1 .18 0.170 0 .130 1 .16 0.173 0 .143 1 .21 0.179 0 .145 1 .20 0 .172 0 . 146 1 .32 0.242 0 .149 1 .07 0.148 0 .141 1 .15 0.166 0 .131 Appendix L (continued) STANDARD DEVIATION TRT ZN MN ug g-X 1 5.2 282 2 4.7 217 3 5.7 573 4 3.1 592 5 4.2 651 6 4.4 881 7 3.5 574 8 2.8 618 9 1.8 233 10 2.5 523 11 3.5 632 12 3.2 45 4 13 2.9 683 14 4.9 550 15 4.5 656 16 2.6 344 17 3.9 382 18 4.6 568 19 5.1 577 20 3.5 231 21 2.7 462 22 2.9 418 23 2.3 383 24 3.6 571 25 3.6 666 259 N P MG cg g 0 .28 0.037 0.026 0 .25 0.028 0.036 0 .23 0.044 0.021 0 .16 0.029 0.015 0 .20 0.038 0.018 0 .12 0.035 0.027 0 .26 0.030 0.037 0 .18 0.066 0 .026 0 .16 0.037 0.016 0 .23 0.051 0 .031 0 .28 0.053 0.028 0 .22 0.047 0.025 0 .17 0.041 0.026 0 .24 0.059 0.018 0 .19 0.049 0.029 0 .19 0.047 0.031 0 .19 0.053 0.026 0 .28 0.043 0.026 0 .22 0.027 0.024 0 .24 0.054 0.022 0 .12 0.037 0.027 0 .27 0.048 0.040 0 .15 0.037 0.040 0 .22 0.032 0.028 0 .17 0.062 0.024 260 Appendix L (continued) NUMBER OF CASES TRT ZN MN N P MG 1 10 10 10 10 10 2 8 8 8 8 8 3 9 9 9 9 9 4 10 10 10 10 10 5 10 10 10 10 10 6 10 10 10 10 10 7 10 10 10 10 10 8 10 10 10 10 10 9 10 10 10 10 10 10 9 9 9 9 9 11 10 10 10 10 10 12 10 10 10 10 10 13 10 10 10 10 10 14 9 10 10 10 10 15 10 10 10 10 10 16 10 10 10 10 10 17 10 10 10 10 10 18 10 10 10 10 10 19 10 10 10 10 10 20 9 9 9 9 9 21 10 10 10 10 10 22 10 10 10 10 10 23 9 9 9 9 9 24 10 10 10 10 10 25 10 10 10 10 10 Appendix L (continued) MEAN FE CU FLWTBR BI3 HTI3 HTI2 Ug g-1  S 42.1 3.1 0.268 1.30 1.76 1.08 44.6 3.4 0.393 1 .40 1 . 78 1.38 41.7 3.2 0.258 1.15 1.63 1.17 43 .2 3.5 0.317 1.22 1.74 1.82 40.7 3.4 0.340 1.17 2.03 1.52 42 .5 3.6 0.316 1.11 1.50 1.74 38.9 3.6 0.366 1.35 1.31 1.58 38.9 3.6 0.257 1.59 2.09 1.35 37.1 3.1 0.272 1.23 1.64 1.12 38.9 3.3 0.291 1.14 1.63 1 .24 44.6 3.7 0.326 1.34 1.43 1.69 41.4 3.2 0.314 1.42 1.46 1.27 38.6 3.4 0.306 1.91 2.36 1.02 42.8 3.4 0 .292 1.60 2.37 0.98 43.2 3.5 0.314 1.77 2.68 0.64 41.8 3.0 0.283 1.60 1.73 1. 70 38.9 3.3 0.223 1.37 2.22 0.78 42 .5 3.5 0.278 1.20 1.43 1.09 45 .0 3.5 0.301 1.38 1.75 1.16 40.9 3.3 0.308 1 .29 2 .06 1 .03 44.3 3.5 0.272 1.41 1.94 1.28 42 .1 3.2 0.307 1.25 1.26 1.03 53.7 3.6 0.742 1.94 1.82 1.49 38.9 3.0 0.233 1.53 2.06 1 .08 52.7 3.9 0.288 1.39 1.69 1.25 Appendix L (continued) STANDARD DEVIATION FE CU FLWTBR BI3 HTI3 HTI2 PS g-X S 7.3 0.7 0.181 0.47 0.88 0.58 8.3 0.8 0.222 0.44 1.04 0.91 3.6 0.4 0.121 0.42 1.41 0.98 7.0 0.6 0.089 0.30 0.44 0.76 20.6 0.9 0.157 0.38 1.26 0.71 12.0 0.8 0.103 0 .22 0.41 0.53 9.3 1.0 0.217 0.36 0.46 0.89 9.4 1.2 0.136 0.95 1.33 0.64 7.7 0.9 0.108 0.35 0.69 0.43 5.5 0.9 0.156 0 .42 0.59 0.46 6.8 1.3 0.152 0.20 0.28 0.87 7.9 1.2 0 .180 0.69 0.86 0.57 5.0 1.1 0.152 0.89 1.28 0.56 9.7 0.5 0.104 0.54 1.20 0.26 8.3 0.8 0.170 0.43 1.56 0.30 11.7 0.8 0.126 1.14 0.48 0.94 11.8 0.9 0.083 0.45 1.49 0.32 8.5 1.3 0.110 0.29 0.46 0.46 8.4 1.0 0.151 0.41 0.56 0.67 6.0 0.6 0.165 0.54 1.09 0.41 10.3 0.8 0.081 0.42 0.98 0.47 6.0 0.7 0.193 0.27 0.67 0.36 7.0 0.7 0.306 1.11 0.83 0.48 7.8 0.7 0.116 0.79 1.14 0.43 12.0 0.9 0.140 0.14 0.56 0.61 263 Appendix L (concluded) NUMBER OF CASES FE CU FLWTBR BI3 HTI3 HTI2 10 10 10 10 to to 8 8 8 8 8 8 9 9 9 9 9 9 10 10 10 10 10 10 10 10 10 to to 10 10 10 10 10 10 10 10 10 to 10 10 10 10 10 10 10 10 10 10 10 to 10 10 10 9 9 9 9 9 9 10 10 to 10 10 10 10 10 10 10 10 10 10 10 10 to to 10 10 10 10 10 10 10 10 10 to to to 10 10 10 10 to 10 10 10 10 to to to 10 10 10 10 10 10 10 to 10 10 10 to 10 9 9 9 9 9 9 10 10 10 10 to to 10 10 10 10 10 10 9 9 9 9 9 9 10 10 10 10 10 10 10 10 10 to 10 to Symbols SOS = sulphate-sulphur TOTS = total sulphur FLWTBR = foliar mass (g) per shoot of the current year's growth Bl = shoot increment ratio HTI = height increment ratio 1 = growth increment in 1985/growth increment in 1984 2 = growth increment in 1986/growth increment in 1985 3 = growth increment in 1987/growth increment in 1986 264 APPENDIX M. FOLIAR NUTRIENT DATA FOR ZINC AND AND AGE. Appendix Ml. Site 1. MEAN TRT ZN1 MN1 ZN2 3 11.0 2971 24.4 4 13.6 1352 48.7 5 15.4 1143 135.5 7 11.5 1231 19.8 8 11.0 1228 17.9 11 8.8 992 13.0 12 9.7 1139 18.5 STANDARD DEVIATION TRT ZN1 MN1 ZN2 3 4.6 1278 5.7 4 2.3 650 20.2 5 5.7 291 40.7 7 3.5 352 6.6 8 3.7 460 4.5 11 2.3 200 2.0 12 3.2 395 11.8 STANDARD ERROR TRT ZN1 MN1 ZN2 3 1.6 452 2.1 4 1.0 265 7.6 5 2.2 110 15.4 7 1.2 124 2.5 8 1.3 163 1.6 11 0.8 67 0.7 12 l.l 132 3.7 NUMBER OF CASES TRT ZN1 MN1 ZN2 3 8 8 7 4 6 65 7 7 7 7 8 88 8 8 8 11 9 9 9 12 9 9 10 MANGANESE (ug g-1) WITH TIME ZN3 MN2 MN3 10.4 4610 3059 51.2 1481 1146 .44.4 1851 1200 5.1 1733 1195 2.5 1464 1203 2.1 1382 1067 2.2 1432 1019 ZN3 MN2 MN3 4.9 2001 1320 17.0 850 289 47.0 722 333 3.1 474 331 1.1 630 340 0.8 395 334 1.4 594 311 ZN3 MN2 MN3 1.8 756 46 7 6.9 321 118 L7.8 273 126 1.2 179 117 0.4 223 120 0.3 132 111 0.5 188 98 ZN3 MN2 MN3 7 7 8 6 7 6 7 7 7 7 7 8 7 8 8 6 9 9 8 10 10 265 Appendix M2. Site 2. MEAN TRT ZN1 MN1 ZN2 ZN3 MN2 MN3 3 12.4 3875 15 .5 3.2 5920 3196 4 11.5 1527 35 .2 53.0 1704 1676 5 19.6 1141 78.7 139.6 1687 1251 7 8.8 1728 15.6 2.0 1082 1633 8 11.4 1181 17.0 2.5 1617 1037 11 10.1 1249 9.5 2.0 1726 1048 12 11.1 1932 14.8 3.0 2365 1474 STANDARD DEVIATION TRT ZN1 MN1 ZN2 ZN3 MN2 MN3 3 5.6 1429 5.7 3.0 2804 1495 4 5.6 1031 18.8 21.9 1322 558 5 8.8 434 34.2 746 101 7 3.4 1025 3.0 0.0 1030 482 8 2.7 601 7.0 2.1 737 677 11 4.2 357 3.5 1.7 206 175 12 4.8 589 8.2 2.2 956 666 STANDARD ERROR TRT ZN1 MN1 ZN2 ZN3 MN2 MN3 3 1.9 476 2.0 1.3 991 498 4 2.1 390 8.4 9.8 591 249 5 3.9 194 15 .3 334 71 7 1.7 513 2.1 0.0 728 278 8 1.1 245 3.1 1.5 330 276 11 2.1 179 2.0 1.0 119 87 12 1.7 208 2.9 1.1 338 222 NUMBER OF CASES TRT ZN1 MN1 ZN2 ZN3 MN2 MN3 3 9 9 8 5 8 9 4 7 7 5 5 5 5 5 5 5 5 1 5 2 7 4 4 2 2 2 3 8 6 6 5 2 5 6 11 4 4 3 3 3 4 12 8 8 8 4 8 9 1 = one-year old foliage formed and collected in 1986, two years after treatment. 2 = two-year-old foliage formed in 1985 and collected in 1986, two years after treatment. 3 = one-year-old foliage formed and collected in 1985, one year after treatment. Equations from Appendix D were used to convert the original AA values to their equivalent values for the ICP which are presented here. 266 APPENDIX N. SCATTER PLOTS OF HEIGHT INCREMENT VERSUS FOLIAR ZINC. Appendix N.l. Scatter plot of first year total height increment (cm) (in 1986) versus first year foliar zinc levels (ug g_t) (for cases where Zn <100 pg g-1) on site 5. 267 Appendix N.2. Scatter plot of second year total height increment (cm) (in 1987) versus second year foliar zinc levels (ug g-M on site 5 . 268 APPENDIX 0. SCATTER PLOTS OF FOLIAR NITROGEN VERSUS FOLIAR ZINC. 1.80 ~z 1.60 z LO = j.„ n 1 craHer ulot of foliar nitrogen (cg g"1) versus tn"r"i2;l;MS""".«'c.:.. where Z. 130 ps f.r c-nt year's foliage (in 1985) from site 2. 269 1.6 -i CD 1.4 i CO CD C 1.2 i CD CD O 1.0 H O "5 0.8 y = 0.8 + 0.038x - 0.0009x R = 0.21 0.6 i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i II i i i i i i | 0.0 10.0 20.0 30.0 40.0 Foliar Zinc 1986 Appendix 0.2. Scatter plot of foliar nitrogen (eg g_1) versus foliar zinc (pg g-M using all treatments (for cases where Zn <30 pg g-1-) for current year's foliage (in 1986 ) from site 2. 1.8 -2 Appendix 0.3. Scatter plot of foliar nitrogen (cg g_1) versus foliar zinc (pg g-1-) using all treatments for current year's foliage (in 1986) from site 3. 271 2.0 n CD 1.6 -3 OO C 1.2 i CD cn O 0.8 H ~0 0.4 ^ l_L_ 0.0 y = 0.137x - 0.0036Lx R = 0.46 I | | | I I I I I I I I I I I I I I I I I I I I I M I | I I I I M I I I | I I I I I I I I I | M I I I I I I I | 00 5 0 10.0 15.0 20.0 25.0 30.0 Foliar Zinc 1986 Appendix 0.4. Scatter plot of foliar nitrogen (cg g_t) versus foliar zinc (pg g_t) (for cases where Zn <30 pg g-1) for current year's foliage (in 1986) from site 4. 272 Appendix 0.5. Scatter plot of foliar nitrogen (cg g-1) versus foliar zinc (pg g-1) using all treatments of current year's foliage (in 1987) from site 4. Appendix 0.6. Scatter plot of foliar nitrogen (eg g-1) foliar zinc (ug g-M (for cases where Zn <40 pg g_1) of year's foliage (in 1986) from site 5. 274 Appendix 0.7. Scatter plot of foliar nitrogen (cg g_1) versus foliar zinc (pg g-M using all treatments of current year's foliage (in 1987) from site 5. 

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