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An investigation into the factors contributing to the growth-check of conifer regeneration on Northern… Montigny, Louise E. M. de 1992

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AN INVESTIGATION INTO THE FACTORS CONTRIBUTING TO THE GROWTH-CHECK OF CONIFER REGENERATION ON NORTHERN VANCOUVER ISLAND  by  LOUISE de MONTIGNY B.S.F., University of British Columbia, 1983 M.F.S., Yale School of Forestry, 1985  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Forest Sciences)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 1992 Louise de Montigny,  1992  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  tJJ.  JtZd2i  The University of British Columbia Vancouver, Canada  Date  DE-6 (2188)  0 J L/(,  79’2  Abstract  Conifer plantations established on cutovers in the CWHb1 zone on Northern Vancouver Island grow well initially, but coincident with the reinvasion of salal Pursh.)  (Gaultheria shallon  on sites formerly dominated by western red cedar and  western hemlock (CH phase) growth stagnates.  the trees become chiorotic and  These symptoms are not seen on sites  dominated by western hemlock and amabalis fir (HA phase). This study examined some site and soil chemical factors which could be responsible for differences in forest productivity by:  1)  phases;  documenting physical differences between the CH and HA 2)  documenting morphological and chemical differences  between the organic horizons found in forest floors of CH and C 3 HA phases using classical wet chemistry techniques and ‘ nuclear magnetic resonance (NMR)  spectroscopy; 3)  determining  seasonal trends in free phenolic acid concentrations of soils under salal on CH cutovers by high speed centrifugation and High Performance Liquid Chromatography, and 4)  determining if  solutions of phenolic acids at field concentrations or of salal leachates have a negative effect on conifer seed phosphorus uptake. germination, growth and short term 32 The HA phase was found to occur on higher topographic  ii  positions making them drier and more susceptible to windthrow events than the CH phase which occur on adjacent, positions.  lower slope  This windthrow process appears to rejuvenate the  site by mixing organic horizons with mineral soil,  increasing  aeration, breaking hardpans, and generally improving site conditions. Six distinct humus horizons were identified on the basis of origin and on degree of decomposition.  The proportion of  humus horizon types on the CH reflected ecosystem maturity and lack of disturbance; that of the HA indicated repetitive windthrow events.  The CH humus horizons tended to have higher  concentrations of K,  Ca, Mn,  and labile polysaccharides,  available S,  lipids, and total  as well as a higher pH.  The HA  humus horizons were found to be higher in available N and P and tended to have a lower C/N ratio for the more well— humified horizons.  Tannin signals from ‘ C NMR spectroscopy 3  were found in the Fm horizons of both CH and HA, but the intensity was greater for the CH.  The source of tannins  appears to be salal, as strong tannin peaks were identified in salal roots,  leaves, flowers, berries and litter.  Tannins are  known to inhibit decomposition and mineralization processes. Concentrations of phenolic acids originating from angiosperms  (presumably salal) were significantly higher in  summer months,  coincident with greater physiological activity  of salal. Phenolic acids are known to cause root membrane dysfunctioning in some situations. iii  The germination values of  seeds and the biomass of seedlings of Sitka spruce, western hemlock and western red cedar tended to be lower given treatments of either a phenolic acid solution at field concentrations or a salal leachate solution compared to a control of distilled water.  The uptake of P 32 by the excised  roots of Sitka spruce and western hemlock were higher for the phenolic acid and salal leachate solutions than for the control, weeks.  indicating P was probably limiting even after only 12 Uptake of 32 P by fine roots of mature western red cedar  and western hemlock was significantly reduced by the phenolic acid and salal leachate solutions. In conclusion, this study provides evidence to suggest that the growth—check of conifer regeneration involves a number of interacting factors.  These include:  1)  the presence  of hardpans and compacted mineral soils which contribute to annual periods of anaerobic soil conditions; 2) of large pockets of nutrient poor woody humus, presence of tannins,  the presence and 3)  the  lipids and phenolic acids in active humus  horizons which may be contributing to decreased decomposition, mineralization and nutrient uptake in trees.  iv  Table of Contents ii  Abstract Table of Contents  v  ListofTables List of Pigi.ires  xi  Acknowledgements INTRODUCTION  CHAPTER I.  1.0 2.0  3.0  3.0 4.0  1  THE PROBLEM PRELIMINARY APPROACHES AND DIFFICULTIES 2.1 Choosing to work with phenolic acids 2.2 Monitoring the release of phenolic acids using XAD Resin . . 2.3 Sampling Forest Floor by Bulk Sampling 2.4 Extraction and Characterization ofPolyphenolics FINAL APPROACH AND HYPOTHESES Study Objectives 3.1 3.2 Overall Hypothesis 3.3 Individual Hypotheses 3.3.1 Soil classification and characterization of CH and HA sites 3.3.2 Chemical characterization of humus horizons using carbon 13 nuclear magnetic resonance 3.3.3 Allelopathic potential of salal  CHAPTER II.  1.0 2.0  xiii  .  .  THE EFFECTS OF ERICACEOUS PLANTS ON FOREST . . . . PRODUCTIVITY: A LITERATURE REVIEW  INTRODUCTION DISTRIBUTION OF ERICACEOUS PLANTS IN THE NORTHERN HEMISPHERE THE HEATHER CHECK SYNDROME EFFECTS OF ERICACEOUS PLANTS 4.1 Allelopathy v  1 4 5 5 10 11 12 12 13 13  13  14 14  15 15 15 18 21 21  50 6.0  4.2 Soil Acidification and Paludifjcation 4.3 Humus Decomposition MANAGEMENT OF SITES DOMINATED BY ERICACEOUS SPECIES StJI.1IIA..Ry  CHAPTER III.  1.0 2.0  3.0  4.0 5.0  SOIL CLASSIFICATION AND CHARACTERIZATION OF CH AND HA SITES  INTRODUCTION METHODS Soil and Vegetation Description 2.1 and Collection 2.2 Laboratory Methods 2.2.1 Nutrient analysis 2.2.2 Polysaccharides and cellulose Lipids 2.2.3 Bound phenolic acids 2.2.4 2.2.5 Statistical analysis RESULTS AND DISCUSSION 3.1 Site Description 3.2 Humus Classification and Variability 3.2.1 Humushorizons 3.2.2 Humusprofjles 3.2.3 Soil variability 3.3 Nutrient Concentration of Humus Horizons 3.3.1 Nutrient concentration of • woody horizons 3.3.2 Nutrient concentration of • non—woody horizons 3.3.3 Nutrient concentration differences • between sites 3.4 Organic Composition of Humus Horizons • 3.4.1 Lipids • 3.4.2 Polysaccharides and cellulose • Bound phenolic acids 3.4.3 • SUMMARY • CONCLUSION • Soil Classification 5.1 5.2 Chemical Differences in Humus Horizons between CH and HA  vi  27 29  •  .  •  .  •  •  31 33  •  .  35  • •  35 36  • • • • • • • • • •  36 38 38 39 39 40 41 41 41 45 45 47 55 61  .  •  61  •  •  63  .  •  •  •  .  •  •  •  •  •  •  •  •  •  64 77 77 81 84 90 95 95 96  CHAPTER IV.  1.0 2.0  3.0  4.0  5.0  CHAPTER V. 1.0 2.0  3.0  4.0  CHEMICAL CHARACTERIZATION OF HUMUS HORIZONS USING CARBON-13 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY  .  INTRODUCTION METHODS Sample Preparation 2.1 2.2 NMR Spectroscopy 2.3 Spectral Analysis RESULTS AND DISCUSSION 3.1 Carbon-13 CPMAS NNR Characterization of Humus Horizons 3.1.1 Woody horizons 3.1.2 Non—woody horizons Dipolar-dephased Carbon-13 CPMAS 3.2 NMR Characterization of Humus Horizons 3.3 Carbon-13 CPMAS NNR Characterization of Litter Inputs 3.4 Site Differences SUIIIvIA.IY CONCLUSION  .  .  .  •  .  .  •  .  •  .  .  •  •  .  •  .  .  •  •  •  .  •  .  vii  97 98 98 98 99 100  100 101 107  110  114 117 119 120  ALLELOPATHIC POTENTIAL OF SALAL INTRODUCTION METHODS Seasonal Phenolic Acid Concentration 2.1 2.1.1 Soil sampling 2.1.2 Sample preparation 2.1.3 HPLC analysis 2.1.4 Statistical analysis • • 2.2 Effects of Phenolics on Conifers 2.2.1 Seed germination 2.2.2 Seedling growing conditions Seedling root bioassay . 2.2.3 2.2.4 Mature root bioassay . . 2.2.5 Statistical analyses • . RESULTS AND DISCUSSION 3.1 Seasonal Phenolic Acid Concentration Effects of Phenolics on Conifers . 3.2 3.2.1 Seed germination 3.2.2 Seedling growth 3.2.3 Seedling root bioassay . 3.2.4 Mature root bioassay StJ1M.ARY  97  121 •  .  •  •  .  .  •  .  .  •  .  .  •  •  .  •  .  .  •  .  .  •  •  .  •  •  •  •  •  .  •  .  .  •  .  .  •  .  •  .  .  •  .  .  •  •  .  •  •  •  •  •  •  •  •  •  121 123 123 123 124 126 127 128 128 130 131 132 133 134 134 142 142 146 147 150 153  5.0  CONCLUSION Seasonal Phenolic Acid Concentrations 5.1 UnderSalal Effects of Phenolics on Conifers 5.2  155 155 156  CHAPTER VI.  OVERALL DISCUSSION  157  CHAPTER VII.  SUMMARY MID CONCLUSION  173  1.0 2.0  173 178  CONCLUSION  L ITERTURE CITED  180  viii  List of Tables CHAPTER III 3.1.  Site Location and characteristics  3.2  Vegetation (% presence and % cover on those plots where present) by stand number  44  Physical descriptions of organic soil horizons on CH and HA sites  46  Proportion of humus forms on CH and HA trench profiles  54  Site property values including mean, standard deviation and occurrence (% of plots) on the CII and HA sites  56  Forest floor horizon depths including mean, standard deviation and occurrence (%)bysite  58  3.3.  3.4.  3.5.  3.6.  3.7.  3.8.  3.9.  Mineral soil horizon depth (cm), standard deviation, and occurrence plots) by site type  .  .  (%  42  .  of .  .  60  Mean nutrient concentrations and standard deviation by horizon on oven— dry and ash—free basis  62  Mean nutrient concentrations, standard deviation and number of samples by horizon and site on oven—dry and ash— free basis  66  3.10. ANOVA for Horizon and Site (Probability of Null Hypothesis) and Squared Multiple R  72  3.11. Organic composition of humus types including lipids, total polysaccharides, labile polysaccharides and cellulose by site based on 4 replicates  78  3.12. 3.13  Concentration of bound phenolic acids (standard deviation) by humus horizon Concentration of bound phenolic acids deviation) by humus horizon and site ix  .  86  (standard 88  3.14.  Pearson correlation matrix between bound phenolic acid concentration and abundance of salal or other shrubs, presence of wood, and site (CH or HA)  89  CHAPTER IV 4.1.  4.2.  Relative percentages of carbon in chemical shift regions of horizons by site Ratios of calculated total lignin, carbohydrate and aromatic carbon and associated ratios for horizons by site  .  .  103  104  CHAPTER V 5.1.  5.2. 5.3.  5.4.  Seasonal pH, water acid concentration deviation by month woody Fm and woody  content and phenolic means and standard and horizon (non— Hw)  135  Germination indices by species, pretreatment and treatment  143  Seedling, shoot, root and total biomass, and uptake of inorganic P by species and treatment  148  Root biomass and uptake of inorganic phosphorus in nanomoles per oven—dry g of mature root  152  x  List of Figures CHAPTER I 1.1.  Continuous root exudate trapping system.  6  1.2.  Soil coring tool  8  CHAPTER III 3.la.  CH trench profile from 0 to 6 in  3.lb.  CH trench profile from 6 to 11  3.lc.  CH trench profile from 11 to 16  3.2a.  HA trench profile from 0 to 4  3.2b.  HA trench profile from 4 to 9  3.2c.  HA trench profile from 9 to 14  3.3.  a) pH, and b) Water content of Humus horizonsbysite  68  a) Total carbon concentration, and b) carbon/nitrogen ratio of humus horizons by site  69  a) Total nitrogen concentration, and b) total sulphur concentration of humas horizons by site  70  Available nutrient concentrations of humus by site including a) available nitrogen, and b) available phosphorus  71  Available sulphur concentration of humus horizons by site  74  Exchangeable cation concentrations of humus horizons by site including a) calcium and b) pottassium  75  a) Exchangeable magnesium, and b) exchangeable manganese of humus horizons by site  76  Concentration of lipids in humus horizonsbysite  79  3.4.  3.5.  3.6.  3.7.  3.8.  3.9.  3.10.  xi  48 49  in in  50  in  .  51  in  .  52  in  53  3.11.  3.12.  a) Total polysaccharides, and b) labile polysaccharides by humus horizons by site  82  Cellulose concentration by humushorizonandsite  83  CHAPTER IV 4.1.  4.2.  4.3.  4.4.  4.5.  4.6.  4.7.  Structural units of a) cellulose, lignin and c) condensed tannins .  b) .  .  .  102  Carbon-13 CPMAS NNR spectra of woody horizons (Fw, Hrw, Hdw) from CH and HA sites  105  Carbon-13 CPMAS NNR spectra of nonwoody horizons (Fm, Hh, IIhi) from CII andHAsites  108  Dipolar dephased Carbon—13 CPMAS spectra of woody horizons (Fw, Hrw, Hw) from CII and HA sites  112  Dipolar dephased Carbon—13 CPMAS spectra of non—woody horizons (Fm, Hh, Hhi) from CII and HA sites  113  Carbon—13 CPMAS NNR spectra of litter materials from a) CH site salal litter, b) CII site coniferous litter and C) HA site coniferous litter . .  115  Carbon-13 CPMAS NNR spectra of salal: a) flowers, b) leaves, and c) roots .  116  Design of soil centrifuge tubes modified from a 30 cc syringe  125  Seasonal phenolic acid concentration (ng/g o.d. soil) in Hw horizons under salal in cutovers  137  Seasonal phenolic acid concentration (ng/g o.d. soil) in Fm horizons under salal  138  CHAPTER V 5.1.  5.2.  5.3.  xii  Acknowledgements I am grateful to Drs. Gordon Weetman, Lawrence Lowe, Karel Klinka, Edith Camm and Morag McDonald of U.B.C. for all their helpful advice, and to Carol Dyck for technical support. Thank—you to Western Forest Products and especially to Dr. John Barker, Bill Dumont, Steven Joyce and Heather Jones without whose field expertise this project would not have been possible. I am particularly indebted to Dr. Caroline Preston for allowing me the opportunity to work at Pacific Forestry Centre and to all the helpful technical advice there from Kevin McCullough, Ann Van Niekerk, Doug Taylor and Rob Hagel. Finally, a very special thank-you to members of my family including Pol, Pierette, Pierre and Denis de Montigny and Raoul Wiart for the many hours contributed to tedious tasks. This work was supported by the South Moresby Replacement Fund (SMRF), the Salal Cedar-Hemlock Integrated Research Project (SCHIRP) and the B.C. Science Council Graduate Engineering and Technology Award (GREAT).  xiii  Chapter I  Introduction  1.0  THE PROBLEM The growth—check of conifer regeneration on northern  Vancouver Island cutovers has been a problem intriguing foresters and researchers on the north coast of British Columbia since the mid 1970’s. sitchensis  At that time, plantations of Sitka spruce (Picea (Bong.)Carr.)  and naturally regenerated western  hemlock (Tsuga heterophylla (Raf.) (Thuja plicata Donn ex D.  Sarge)  and western red cedar  Donn), which were established in the  late 1960’s and which had initially grown well, with leader growth up to 50 cm, began to show signs of stress.  The annual  leader growth was 5 to 10 cm and the needles displayed chiorosis, suggestive of nitrogen and phosphorus deficiencies (Weetman l989a,b).  Coincident with the plantation “check” was the  reinvasion of the cutovers with salal.  1  Trees planted on  roadsides or landings, or those growing on adjacent cutovers without salal, had leader growth of 0.5  in  and no apparent  chlorosis. Lewis  (1982)  the Thula plicata  classified the different sites as “phases” of -  Tsuga heterophylla  Rhytidiadeiphus loreus or “salal  —  -  moss”  Gaultheria shallon  -  Within this  ecosystem.  single ecosystem association, the phases represented two very different kinds of forest occurring side by side: the cedarhemlock (CH) phase being the climatic climax community, hemlock—amabalis fir (HA) sites  and the  phase being a seral stage occurring on  with a history of soil disturbance. The CH phase consisted of somewhat open western red cedar  and western hemlock stands with a minor component of amabalis fir and a dense understorey of salal.  These stands were believed to  have been left undisturbed for as long as 1000 years.  Following  clearcutting, with or without slashburning, the secondary vegetation was overwhelmingly dominated by salal.  Estimates of  above—ground biomass of salal were found to increase from 1058 kg 1 two years after cutting and burning, to 4078 kg ha ha t after 8 years  (Messier and Kimmins,  1990).  The growth-check of conifer  regeneration occurred on these CH cutovers. The HA phase occurred on sites with a history of site disturbance,  such as fire, windthrow,  or on steeper terrain where  soil creep and periodic windfall disturbed the soil. The resultant stands consisted of even—aged, densely stocked western hemlock and amabalis fir, with relatively clean forest floors 2  dominated by mosses.  Shrub and herb cover were sparse and tended  to favour mineral soil overturns.  Planted and natural  regeneration did not show the same symptoms of growth—check and foliar nutrient deficiencies.  The occurrence of communities  intermediate between the climatic climax CH phase and the seral HA phase lead Lewis  (1982)  to state that the HA phase had the  potential to develop into the CH phase, given a sufficiently long period of time without soil disturbance.  Part of the focus of  this study was therefore to examine some of the chemical and physical site differences between the CH and HA phases to determine if these factors may be,  in part, responsible for the  growth-check of conifer regeneration on the CH but not the HA phase. The differences in productivity between cutovers formerly occupied by salal—rich cedar—hemlock sites and adjacent salal— free hemlock—amabalis stands have been thought to be due to competition by salal.  Attempts to eradicate in order to reduce  the level of competition by salal have proven to be very difficult.  The thick cuticular wax and deeply rooted,  easily  propagated rhizome of salal have made eradication by herbicides, fire,  scarification, or clipping almost impossible.  Attempts to  ameliorate the nutrient deficiencies of the trees by nitrogen and phosphorus fertilization have been temporarily successful.  The  restoration of rapid cedar growth lasted 5 to 7 years but foliar nutrient concentrations soon returned to pre—fertilization deficiency levels.  The extent of the problem quickly became 3  evident, as an estimated 100,000 hectares of these salal ecosystems are found throughout the wetter portions of the Coastal Western Hemlock biogeoclimatic zone on Vancouver Island, the coastal mainland and the Queen Charlotte Islands. Experiences in the United Kingdom with Sitka spruce planted in dense swards of heather (Calluna vulgaris  (L.) Hull)  brought  some interesting ecological parallels to light: salal and heather are both ericaceous shrubs, the symptoms of growth—check including slowed growth and chlorosis are identical, and the rapid but temporary response to fertilization is similar (Malcolm,  1987).  in Chapter 2)  Evidence for allelopathy by heather (discussed  has been accumulating, and the question naturally  arose whether salal too, could be allelopathic.  To date,  little  work has been done on the allelopathic role of salal in these coniferous ecosystems.  Part of the focus of this study was  therefore, to determine if salal could be responsible for the production of compounds which could have an allelopathic effect on conifers.  2.0  PRELIMINARY APPROACHES AND DIFFICULTIES This project was intended to be an exploratory study  examining some of the physical site factors which may be responsible for differences in conifer regeneration after cutting, and the possible role of salal as an allelopathic competitor to conifer species in plantations.  It was intended  from the start that results which proved promising would be  4  pursued, while those that failed would be abandoned.  The  following describes some initial work which was felt to be inappropriate for the study,  2.1  and reasons why they were abandoned.  choosing to Work with Phenolic Acids  Much of the literature dealing with allelopathic activity of ericaceous plants suggests that both phenolic acids and fatty acids may be the principal allelochemicals involved in alleged conifer—ericaceous allelopathic interaction.  However, a gas  chromatograph, which would have allowed an examination of fatty acids in these ecosystems, was not available during the course of this study.  It was therefore decided to limit the study to that  of phenolic acids.  2.2 Monitoring the Release of Phenolic Acids Using XAD Resin  The state—of—the—art method for examining root exudation of phenolic acids was the Continuous Root Exudate Trapping System (CRETS)  described by Tang and Young  (1982),  shown in Figure 1.1.  Salal was grown in acid-washed sand in inverted 2.5 litre glass bottles with the bottom removed.  A very dilute nutrient solution  was recycled through the pot using a continuous air stream, and the solution passed through a column containing XAD-4 resin which absorbs hydrophobic and partially hydrophobic compounds, phenolic acids.  such as  The accumulation of root exudate components  released over a period of time could then be detected, produced at very low levels.  5  even if  PLANT----  -  ROCk SOLIJTI ON COARSE  Al R TEFLON  - - -  TEFLON SLEEVE  GLASS WOOL TEFLON SLEEVE CONNECTION  Figure 1.1:  Continuous root exudate trapping system (From Tang and Young, 1982). 6  Initial analysis of phenolic acids desorbed from the XAD-4 resin indicated large concentrations of one acid, but this was attributed to resin degradation.  After more effective clean—up,  trace amounts of some phenolic acids were detected, but the most polar compounds were absent. quantitative estimates.  Levels were too low to allow  It was assumed that the results had  demonstrated that salal did not exude phenolic acids.  A similar  approach was used to study variability of phenolic acids in soil solution of forest humus.  A coring tool was constructed in which  a stainless steel cylinder could cut and capture a soil core within a PVC cylinder, which could then be capped for transfer and storage, then easily modified to act as a leaching tube without any further transfer or disturbance of the soil core (Figure 1.2).  A total of two hundred cored samples were  collected and leached through XAD-4 resin at a field laboratory set up at Port McNeill.  The phenolic acids were desorbed using  methanol and kept refrigerated until analyzed through HPLC at U.B.C. Measurement of seven separate phenolic acids showed no systematic relationship to soil phase (CH or HA). Protocatechuic, p—hydroxybenzoic, and vanillic acids were commonly detected, although at rather low concentrations. and syringic acids were sometimes found.  Gallic  Interference by  coloured contaminants, manifested by erratic baselines, made quantification of some acids unreliable.  Some attempts were  influenced by ‘bleeding’ of breakdown products from the XAD-4  7  3ft pusher  —pvc coupler  /nut  core cutter  Figure 1.2:  Soil coring tool.  8  resin.  This was largely overcome by better column clean—up.  The composition of phenolic acids obtained in the CRETS and field study raised questions about the adequacy of recovery of all the compounds expected.  A careful evaluation of the recovery  of standard compounds, using the published XAD-4 method for separating phenolic acids from aqueous solutions, failure of XAD—4  (even under very acid conditions)  indicated the to  quantitatively recover the phenolic acids present.  While the  recovery of the less polar acids like vanillic and ferulic were reasonable,  losses of the most polar acids were excessive (53%  and 89% for protocatechuic and gallic respectively). of other solid adsorbents, used,  Examination  and of variation in pH and eluents  failed to overcome this problem. An additional problem with the resin system lay in the fact  that XAD resin allows simple phenolic compounds to continue recycling with the nutrient solution, and thus renders them susceptible to further microbial degradation.  This makes any  estimates of phenolic acid accumulation rather dubious. In light of the problems noted,  it was concluded that the  XAD approach could not yield reliable quantitative data,  could well yield misleading results.  and  In relation to published  studies of allelopathic effects, a CRETS system can presumably be used to demonstrate the presence of potentially allelopathic agents, but caution must be used in accepting claims for allelopathic effects without rigorous verification of method reliability.  Furthermore,  allelopathic effects shown by exudate 9  fractions, may or may not be due to the specific chemicals analyzed,  2.3  as other active ingredients may also be present.  Sampling Forest Floor by Bulk Sampling  The initial study of free phenolic acids in forest floors using resins was based on a bulk sample of forest floor material cored and kept intact for subsequent leaching.  As previously  described the resin method itself was found to be inadequate, but other deficiencies in the methodology were subsequently recognized. Following leaching of the intact soil cores, Munsell colours, pH, horizon sequence and depths were recorded for all samples.  Although pH rarely varied by more than 0.5 pH units at  any one plot, the proportions of horizon types in each core reflected substantial spatial variability.  In particular the  proportion of decayed wood varied a great deal.  Most cores  contained two distinct horizon types in varying proportions. It was concluded that demonstrating significant differences in phenolic acid levels or composition would require a much more homogenous sample base than that provided by random cores, unless a prohibitively large number of samples were collected. same time,  At the  it was confirmed that rather large sample weights  would be needed for reliable analytical determination of individual phenolic acids by HPLC. A preliminary set of grab samples collected from trench transects at Port McNeill indicated a dichotomy between woody and  10  non—woody samples and within each, a variation in degree of humification.  The differences in plant origin and associated  properties reflect differences in biochemical processes, and even microsite variations within the forest floor. an understanding of these processes,  In order to gain  it was therefore deemed  necessary to study the behaviour of individual types of materials which would be reasonably well characterized.  A further  advantage of working within defined horizons is that of allowing more effective communication with other investigators working on similar  2.4  (or dissimilar) materials.  Extraction and Characterization of Polyphenolics  Published methods used to extract polyphenolics involve shaking overnight in acetone, concentrating, then extracting with ethyl acetate to remove waxes and low molecular weight compounds. The aqueous extract is then freeze—dried, and eluted through a column of Sephadex LH—20 using combinations of methanol, water and acetone.  Carbon—13 Nuclear Magnetic Resonance Spectroscopy  is then used to identify the final product. When this procedure was used, there was a thick suspension between the acetone and ethyl acetate layers, and no clear demarkation between any of the layers, thus making removal of waxes and low molecular weight compounds difficult.  The  resulting extract was concentrated and run on Sephadex LH—20 resin using 1:1 water:methanol, developed on concentrating.  although a thick waxy layer  Three coloured bands were separated:  11  a very fast brownish band; a medium-fast light-yellow band, and a slow, greyish—yellow band. down and freeze dried.  These were individually evaporated  The final extract, presumably containing  the tannins, was removed with 70% acetone, evaporated down, and freeze—dried.  The procedure was replicated 4 times.  When these extracts were examined using NMR, results made no sense,  looking more like degradation products of the Sephadex  than tannins.  It was concluded that the published methods were  poorly described.  An attempt was made to find a suitable method  for tannin extraction, and Waterman  but none was found.  (1987a and b)  Interestingly, Mole  compared several methods for measuring  tannins and concluded none were consistent.  This approach was  therefore considered beyond the scope of this study and was not pursued.  3.0  FINAL APPROACH AND HYPOTHESES 3.1 Study Objectives The final approach of the overall study has two objectives:  Oblective 1: To document in detail some of the differences in soil and stand characteristics between CH and HA phases,  including  morphological and chemical differences between the organic horizons found in forest floor horizons, using classical wet chemistry techniques,  as well as  NMR mass spectrometry.  It was anticipated that this would allow the identification of factors responsible for growth stagnation on CH sites. 12  Obiective 2: To determine seasonal trends in phenolic acid concentrations under vigorous salal on regenerating cutovers on CH sites and to determine if the concentrations found had an allelopathic effect on conifer seed germination,  growth, and  short term phosphorus uptake.  3.2 Overall Hypothesis  The overall hypothesis of the study is that there are differences in chemical characteristics of similar humus horizons between CH and HA sites, and furthermore, these differences may be the cause of the poor productivity of conifer regeneration on cutover CH sites.  3.3  Individual Hypotheses Soil classification and characterization of CH and HA phases  3.3.1.  1)  That distinct and recognizable humus horizons occur commonly on both the CH and HA sites, but that the relative abundance of the horizons varies between the sites,  2)  and  That similar horizons from the CH and HA sites are different with respect to chemical composition including total and/or available nutrient concentrations,  lipid and polysaccharide contents, and  phenolic acid content.  13  3.3.2  1)  Chemical characterization of humus horizons using ‘ C Nuclear Magnetic Resonance 3  That the NNR spectra obtained from the different humus horizons are distinct and recognizable,  2)  and  That similar horizons from the CII and HA sites are different with respect to phenolics and tannins.  3.3.3 1)  Allelopathic potential of salal  That the concentration of free phenolic acids found under salal in plantations will vary with season and will be highest during the summer months when salal is most physiologically active,  2)  and  That solutions using the maximum concentrations of free phenolic acids found in soils under salal and of leachates from the flowers and berries of salal, cause reduced seed germination, biomass growth,  will and 32 P  uptake in roots of Sitka spruce, western hemlock and western red cedar.  14  Chapter II  The Effects Of Ericaceous Plants on Forest Productivity: A Literature Review 1  1.0  INTRODUCTION The chlorosis and stagnation of conifer plantations  associated with salal on northern Vancouver Island was found to be similar to that reported for other conifer—ericaceous dominated ecosystems  (Weetman  p1.,  1989a,b).  This led to the  speculation that perhaps ericaceous plants use similar mechanisms to achieve similar symptoms in conifers.  The literature was then  reviewed to gain an understanding of the history and extent of the problem in other areas of the world and the results of past research dealing with ericaceous plants.  2.0  DISTRIBUTION OF ERICACEOUS PLANTS IN THE NORTHERN HEMISPHERE Ericaceous plants are one of the most ecologically success  ful plants in the Northern Hemisphere, with representatives 1  This chapter is based on the publication by de Montigny and Weetman (1990). 15  dominating vast areas of heathiands.  The  term “heathland” is  used to describe territories in which trees or tall shrubs are sparse or absent, and in which the dominant life—form is that of the ericaceous dwarf shrub,  as represented by the order Ericales,  particularly of the family Ericaceae, which comprises about seventy genera and more than 1900 species.  Specht  (1978)  has  enumerated the conunon traits for worldwide heathland communities as follows:  1) their evergreen scierophyllous nature; 2) the  presence, but not necessarily the dominance, of the heath families in the stand Ericaceae,  —  Diapensiaceae, Empetracaea,  Epacridaceae,  Grubbiaceae, Prionotaceae, Vacciniaceae, and 3) their  ecological restriction to soils very low in plant nutrients. These infertile soils may be well-drained (supporting dry heathlands or  ‘sand—heath’)  or seasonally waterlogged (supporting  wet-heathland). The conditions favouring the dominance of heathlands involve a combination of a relatively cool temperate regime, high humidity throughout most of the year,  and rather freely-drained  soil not conducive to formation of peat.  In addition,  some  factor, whether climatic, edaphic, biotic or anthropogenic, must operate to initially remove, or exclude the development of taller shrubs and trees.  This type of environment may be expected in  three main categories of situations  (Gimingham,  1972):  1) where  for any reason forest is excluded in the strongly oceanic regions of the cool-temperate belt;  2)  in certain parts of sub-arctic  and sub-antarctic territory, and 3) where adequate humidity 16  prevails at sub—alpine or low—alpine altitudes on mountains. Of the three situations, the first is of particular economic and land—use importance in developed countries. Oceanic heathlands are most widely represented in western Europe,  South Africa, Eastern Canada and U.S.A.  In Europe, the  heath region belongs essentially to the oceanic and sub—oceanic regions of west Europe, particularly the broad west European coastal plain in countries bordering the North Sea and the English Channel. Calluna.  The most prominent genera are Erica and  In Canada, the heathlands predominate in Nova Scotia,  Newfoundland and coastal British Columbia, while in U.S.A., mainly along the extreme eastern coastline from Maine to South Carolina and the Alaska panhandle; the prominent genera are Vaccinium, Gaylussacia, Gaultheria, Rhododendron, Kalmia and Arctostaphylos. Heathlands of the cool—temperate oceanic region are believed to have arisen from pre—existing forests following human settlement and land cultivation.  Analytical studies of pollen  indicate the expansion of ericaceous pollen in relation to tree pollen in Denmark (Jonassen,  1950)  and Norway  (Kaland,  1986).  Historical land use studies indicate that repeated cutting and burning for agricultural land use effectively maintained and perpetuated the heath vegetation over considerable areas of Britain (Conway, (Behre,  1986)  1947),  Sweden and Denmark (Romell,  and Newfoundland  (Meades,  1986).  1952), Norway  Similarly,  clearcut logging and slashburning on northern and western 17  Vancouver Island has stimulated the productivity of salal (Gaultheria shallon Pursh.) which appears to be correlated with the exclusion of forest tree regeneration (Weetman 1989a,b).  Salal biomass was found to almost quadruple from 1058  kg ha 1 at 2 years after cutting and burning, to 4078 kg ha’ at 8 years, while below—ground biomass  (including roots of Vaccinium  spp) were found to increase from 1449 kg ha-’ at 2 years to 8507 kg ha-’ at 8 years  3.0  (Messier and Kimmins,  1991).  THE HEATHER CHECK SYNDROME The effect of ericaceous plants on trees was first observed  between Calluna vulgaris sitchensis (Bong.)  Carr.)  (L.)  Hull and Sitka spruce (Picea  in Scotland by Muller (1897) who  commented that although cultivation of the heathiand soil by ploughing and harrowing several times in the course of three years resulted in satisfactory growth of spruce, this was only maintained so long as heather did not re—invade the sites. Prevention of re—invasion by heather allowed the spruce to continue to grow but once the heather covered the site again, stagnation of the spruce ensued; this effect was more pronounced where there had been more rapid growth of the spruce following cultivation of the soil. In early trials of afforestation on the heathiands,  it was  noted that “heather—sensitive” species such as Sitka spruce, Norway spruce (Picea excela Link), menziessii  (Mirb.)  Franco),  Douglas—fir (Pseudotsuga  silver fir (Abies amabalis 18  (Dougi.)  Forbes), western hemlock (Tsuga heterophylla (Raf.) Lawson cypress  (Chamaecyparis lawsoniana  (A. Murr.)  Sarg.), and Pan.),  virtually ceased growth when planted in Calluna swards.  Pioneer  species such as pines and larches, did not suffer this “check” to their growth (Weatherall, 1953). Fertilizer applications led to the belief that stagnation of Sitka spruce on heather—dominated sites was the result of direct competition by heather for water and nutrients, especially nitrogen (N)  and phosphorus  But, the elimination of heather  (P).  did not always alleviate the checked condition, particularly on sites deficient in N and P.  Fertilization of these sites with N  and P resulted in a temporary growth response, but it soon became apparent that further fertilization was necessary.  Thus, the  Calluna check was thought to be caused by more than just a nutrient deficiency (Malcolm, Braathe  (1950)  1975).  suggested unfavourable soil conditions, deficiency of trace elements in the soil,  excessive soil acidity,  deficiency of mycorrhizal fungi and severe competition from Calluna as being responsible for the inhibition of growth of spruce on heathlands.  He found it very difficult to believe that  the soil could change so markedly in two to three years, or that Calluna at the time of invasion, nutrients so completely.  could monopolize all the  He therefore concluded that Calluna  had a biological effect on spruce and suggested that a substance was produced which in some way inhibits the growth of spruce. Circumstantial evidence for allelopathy by the Ericaceae was  19  gathered in Scotland where it was noted that species sensitive to heather competition did not develop the branched mycorrhizal root systems typical of the normal condition of actively growing plants.  This led to the speculation that some factor closely  associated with the Calluna plant prevented the development of the ectomycorrhizal association in the trees.  Handley (1963)  found that aqueous extracts of mor from stands of vigorous Calluna  could inhibit the growth of a range of mycorrhizae  forming fungus, whereas extracts from other soils of similar acidity and nutritional status did not.  Some fungi,  including  those that formed mycorrhizal associations with pine, were not inhibited.  The inhibition was less pronounced where the mor  sample came from shaded Calluna or was less acid.  There seemed  to be some variation in the resistance of fungi to the inhibitory factor,  suggesting that some strains of fungus could form ecto—  mycorrhizal associations with trees such as pines, that could apparently tolerate the Calluna competition.  The disappearance  of the factor inhibitory to the mycorrhizae when the Calluna was suppressed,  suggested that it must be continuously produced to  maintain an inhibitory level. Handley’s work did not achieve immediate acceptance,  and as  late as 1970 researchers were still suggesting direct competition of Calluna was inhibiting the growth of trees  (Bjorkman,  1970).  However, the ability of “heather—sensitive” species to compete with other vegetation,  some of which is more demanding in its  water and nutrient demands than Calluna, would seem to relegate 20  direct competition to a somewhat lesser role. According to Read (1984), toxicity of heathiand soils occurs as a result of the ability of Calluna to modify the soil environment in its favour. acid content of humus,  Interactions between the high organic  low pH,  and low base status produce  phytotoxicity sufficient to exclude or debilitate most would-be competitors.  The success of ericaceous species therefore must be  examined in terms of all interacting effects.  4.0 EFFECTS OF ERICACEOUS PLANTS 4.1  Allelopathy  The word allelopathy was coined by Molisch (1937) describe the chemical interactions among all plants higher plants), influences.  to  (microbes and  including stimulator as well as inhibitory  Typically, and in this case, the word is used to  describe only the harmful effects of one higher plant upon another,  since allelopathy translates literally as “mutual  suffering”  (Putnam and Tang,  1986).  It must be remembered  however, that many cases of allelopathy involve microbes either directly or indirectly,  and some chemicals found to inhibit  growth of the some species at certain concentrations may stimulate the growth of the same or different species at lower concentrations  (Rice,  1984).  Some of the earliest observations of allelopathy concerned harmful effects of crops upon other crops or weeds. (ca.  300 B.C.)  observed that chick pea  21  Theophrastus  (Cicer arientum)  “exhausts” the ground and destroys the weeds. Secundis,  1 A.D.)  not only reported that a number of crops  including chick pea, (Vicea ervilia)  Pliny (Pliniua  barley (Hordeum vulgare), and bitter vetch  “scorch up” cornland, but he also recognized  toxicity of walnut (Julans recfia)  trees.  He attributed the  toxicity of plants to their scents or juices and indicated that bracken fern (Pteridium aguilinum) might even be controlled by breaking young stalks and allowing “the juice trickling down out of the fern to itself kill the roots”. Two likely classes of compounds implicated in allelopathy of ericaceous plants are phenolic and aliphatic compounds. and Williams  (1973)  found that simple phenols were abundant in  the Ericaceae, particularly hydroxybenzoic, vanillic, p—coumaric acid, (1966)  Harborne  cinnamic, gentisic,  and caffeic acids.  Towers  found p-hydroxybenzoic, o-pyrocatechuic, gentisic,  protocatechuic, vanillic,  syringic, p—coumaric,  caffeic,  ferulic  and sinapic acids in hydrolyzates of ethanolic extracts of salal. Of the common flavonols, quercetin is found to occur in all species, while both kaempferol and myrcetin are of more limited occurrence.  Myrcetin is found in more woody members of the  family such as Rhododendroideae,  Ericoideae, Vaccinoideae,  and  Cassiopeae, while kaempferol occurs predominantly in the more herbaceous members such as Pyroloideae and Monotropoideae (Harborne and Williams,  1973).  Aliphatic compounds are also potential allelochemicals because the fat content of ericaceous plants is extremely high, 22  and fatty acids have been shown to be a major lipid storage product (Tschager  1982).  The chain length distribution  of alkanes in the epicuticular wax of many Ericacae and closely associated Epacridaceae vary from C 23 to , 35 with C C 31 the major alkane,  and the odd—carbon chains exceeding the even—carbons  (Salasoo,  1981;  1983a and b; 1987).  The high concentrations of some organic acids in the Ericaceae and/or the allelopathic interaction between ericaceous and non—ericaceous species has been examined for Erica scoparia L. and  . australis L. in Spain (Ballester  Carballeira,1980; Carballeira and Cuervo,  1977; 1980); for  Arctostaphylos glauca Lindl. and A. cflandulosa Lindl. California chaparral (Muller  .,1968; Chou and Muller,  for Calluna vulgaris in Scotland Jalal et al.,  in the  (Handley,  1963; Robinson,  1972); 1972;  1982; Jalal and Read l983a and b); for Kalmia  angustifolia L.  in Newfoundland (Mallik,  1987); and Emetrum  hermaphroditum in Sweden (Zackrisson and Nilsson,  1989).  The occurrence of phenolic or aliphatic compounds in living tissue does not prove an allelopathic effect exists,  or even that  these compounds are released into the soil environment. Furthermore, the release of plant—produced phytotoxins and correlated toxic qualities of the environment does not mean that the original toxic product acts, allelopathic agent.  in unchanged condition,  as the  Some factors of the environment that affect  retention or alteration of allelochemicals include redox potential of the soil,  its fixation on clay or humus, the 23  presence of metallic ions for chelation reactions, and the composition of the soil solution and atmosphere 1983; Haider and Martin,  1975; Huang  (Vaughan  1977).  For example,  levels of phenolic acids have been shown to vary seasonally in soils under Erica australis L. Calluna vulcaris  (Carballeira and Cuervo,  (Jalal and Read,  1983a and b)  1980)  and  ranging from very  low levels in late summer months to maximum levels of 0.12 mM in early summer months.  The seasonal nature of phenolic acid  concentration was felt to be associated with accumulation during cold, wet winter months.  At the outset of spring,  increasing  aeration and temperature lead to more rapid breakdown by increased microbial and fungal growth and metabolism with increased production of phenolics at the roots 1982).  The fatty acids  (Jalal  (hydroxyalkanoic acids) were found in  quantities comparable with or greater than those of the aromatic moieties.  They felt that lipids reaching the soil would  eventually be degraded by the /3 oxidation pathway into decanoic, nonanoic and octanoic acids along with lower fatty acids. The mechanisms by which organic acids act as allelochemicals is still unknown, but some work has been done to determine the likely mechanisms.  Takijima (1964)  found increasing toxicity of  aliphatic acids with increasing carbon chain length accompanied by an increasing affinity for lipids.  Similarly, Glass  (1973)  found the degree of inhibition of ion uptake in roots was correlated well with the lipid solubilities of phenolic acids tested.  Further studies by Glass  24  (1973;  1974;  1975;  1976)  and  Glass and Dunlop  (1974)  showed that a likely mechanism for  reduced ion uptake by phenolic acids is that the phenolic acids partition themselves between the aqueous medium and the lipid component of the cell according to their lipid  solubilities. The  membrane then becomes permeable to both anions and cations.  This  resultant loss of ions rapidly depolarizes the membrane potential by increasing the permeability coefficients of the ions and by reducing the imbalance of ion concentrations across the cell membrane.  The dysfunctioning of the plasma membrane then leads  to the failure of cells to maintain proper mineral nutrition. This could then lead to the inefficiency of the energy systems of respiration and photosynthesis in plants, which demand precise membrane organization, charge separation, and the work of membrane—associated proteins. A somewhat similar effect on growth, dysfunctioning,  as occurs with membrane  could occur with impairment of the establishment  of a mycorrhizal association by trees growing on these nutrient deficient,  acidic sites.  Several studies have shown the  fungitoxic effect of Calluna on mycorrhizal associations of competing species.  Handley’s  (1963)  experiments showed that  Calluna, or its ericoid mycorrhizae, were responsible for the release of a factor inhibitory to the formation of ectotrophic mycorrhizal associations of competing trees.  The prevention of  the mycorrhizal association was due, not to lack of fungal inoculum, fungus.  but to the prevention of growth and inoculation by the Robinson (1972)  confirmed Handley’s observations. 25  Fatty  acids of intermediate chain length (C6-C14)  are potent inhibitors  of growth and respiration of micro—organisms Schillinger,  1944; Wyss  1945).  and mycelial growth are affected.  (Franke and  Both spore germination  The sensitivity of  ectomycorrhizal fungi like Suillus variegetus to octanoic and nonanoic acid (Pederson,  1970) might lead to their exclusion from  soils and explain the widespread inhibition of the fungi reported by Handley (1963). The inability to properly absorb nutrients at the membrane, or to form mycorrhizal associations, can then have far—reaching implications for overall plant growth.  Alterations of the  mineral content of plants subjected to nonspecific allelopathic conditions has been shown in many investigations (Rice,  1984),  but it is difficult to generalize about changes in mineral content incurred from allelopathic interference.  Phosphorus  contents are frequently reduced, while nitrogen, potassium and magnesium uptake may be increased or decreased.  Consistent with  these findings of nutrient imbalance is that tree species “sensitive”  to ericaceous plants often show symptoms of N and P  deficiency (Malcolm,  1975; Weetman  j.,  1989a,b).  Fertilizer  applications have been shown to overcome allelochemical induced growth suppression in laboratory experiments, field experiments using “sensitive” trees  as well as with  (Malcolm,  1975).  Soil or substrate fertility can affect the toxicity and rate of breakdown of phenolics; higher fertility leads to a more rapid breakdown and less toxic conditions.  26  This was shown in a  study by Stowe and Osborn (1980) where phenolic toxicity appeared to depend intimately on nutrient concentrations; the phenolic acids were uniformly and significantly inhibitory only at low nutrient concentrations.  Since phenolics are more likely to be  produced in a plant under stress  (Rice,  1984)  it appears that  allelopathy with phenolics is more likely in nutrient poor soils.  4.2  Soil Acidification and Paludification  Heathland flora is generally indicative of oligotrophic, acidic soils, but more significantly, ericaceous plants may actually contribute to the process of soil acidification, which may be one reason why they are capable of invading and dominating more complex vegetation types. Dimbleby  (1962)  Pollen analysis of heath soils by  has shown that the rise of dominance of Calluna  is closely linked with increasing soil acidification, the disappearance of deep—burrowing earthworms and the subsequent accumulation of raw humus.  Similarly,  Webley  (1952)  have shown marked reductions in bacteria and increases in fungi when a fixed Ammophila sand dune community was succeeded by a dune heath dominated by Calluna.  Grubb  (1969),  found a  strong correlation between the size of Calluna bushes and the soil pH beneath their centres; also between distance from the centre and pH both at the soil surface and below.  More direct  evidence for soil acidification by ericaceous plants was shown in a study with Calluna and Rhododendron, which dominate soils of very low pH in the range 3—4.  When grown in sand with mineral 27  nutrient solution at pH 4.5,  Calluna and Rhododendron acidified  the medium to below pH 4.0 in 8 weeks, and to 3.5 in a subsequent 2 month period (Read,  1984).  The increase in acidity was felt to  be due to production of organic acids, particularly from the oxidation of long-chain fatty acids,  and to the depletion of the  soil base status. The presence of well developed heath vegetation is associated with soil podsolization,  in which sesquioxides are  eluviated to lower mineral horizons  (Soil Survey Staff,  The solubility of iron,  1975).  aluminum and manganese are greater under  the more acidic conditions, and the organic acids associated with heath vegetation then chelate with the metal ions and are leached down the soil profile to the lower horizons. iron-podsols form,  In many cases  in which the iron is deposited in the form of  a thin, hard iron pan (placic horizon or fragipan) consequent rise in the perched water table, widespread paludification (Damman,  leading to the  and ultimately in  1965; McKeague  1968).  The formation of pans under heathiands has been noted in north England and Scotland under Calluna (Gimingham, Newfoundland under Kalmia  (McKeague  Vancouver Island under Gaultheria  .].,  (Carter,  1968), 1988),  southeast Alaska under Vaccinium and Menziesia 1979).  1972),in on western and in  (Ugolini and Mann,  If such sites are left undisturbed, the high water table  can lead to reductions in forest productivity and changes in vegetation through accumulation of forest humus resulting in wetter,  colder soils, and a reduction in tree rooting depths. 28  4.3  Humus Decomposition  As previously discussed,  oxidation products of fatty acids  are fungitoxic and can therefore affect decomposition in soils rich in lipids and other fatty acids.  A further effect of the  apparent accumulation of organic acids under ericaceous plants may be the tanning effect of polyphenolic compounds on humus. Tanning is the process by which proteins are made resistant to decomposition through bonding with a polyphenolic molecule such as hydrolysable tannins condensed tannins  (tannic acid or gallotannins)  (proanthocyanidins).  or  Hydrogen bonds are formed  between the hydroxyl groups of the tannin molecules and the carbonyl groups of the protein—amide linkages and covalent bonds are formed between quinone residues and free amino groups in amino acids.  Both of these reactions modify protein structure.  The efficiency of tanning is associated with the molecular dimensions of the tanning agent,  since the tannin has to form a  stable cross link with the protein molecule.  Tannin molecules  below the critical size cannot form these cross links,  and those  above the size will combine only at easily accessible outer sites producing a case—hardening or surface combination.  It appears  that the tannins formed between pH 3 and 5 are of about the right molecular size to afford adequate protection to the protein. Outside of this pH range,  the molecular size is probably too  great and the protein is not adequately protected 1956).  (Gustaven,  These results suggest that acid conditions, such as those  29  associated with the mor sites, will favour the formation of stable protein—tannin complexes so that mineralization of the protein is delayed.  Since as much as half of total nitrogen (N)  in humic substances can be accounted for as amino acid—N (Stevenson,  1982),  this delay in mineralization may result in N  deficiencies for growing plants. Tannins can also slow the decomposition of nonproteins such as cellulose and hemicellulose because the tannin—protein complexes coat and permeate cell walls, making them considerably resistant to microbial attack (Benoit and Starkey,  1968a and b).  Tannins have been found to be most tightly bound to cellulose at pH 1 to 5, below the isoelectric points of the proteins, and tend to dissociate above pH 5, Starkey,  above the isoelectric point (Benoit and  l968a).  Tannins also affect decomposition through inactivation of certain enzymes important to the process of decomposition of large molecular weight compounds such as proteins, cellulose, hemicellulose and other polysaccharides and lipids. The degree of complexing of an enzyme by tannin will be affected by the chemical properties of the tannins, the ratio of tannin to enzyme, the presence of substances that regenerate the enzyme activity, the composition of the enzyme-protein, and the pH of the liquid in which they are contained (Benoit and Starkey, 1968b) The mor humus forms associated with ericaceous species are characteristically acidic, deep and not conducive to rapid 30  decomposition.  Not surprisingly then, these same sites show very  low rates of mineralization of nitrogen, phosphorus and sometimes sulphur, consequently, there are overall deficiencies of these important nutrients.  It may be then, that the litter of  ericaceous shrubs are rich in polyphenolics and long—chain fatty acids, which when released into the soil greatly reduce the mineralization of organic nitrogen and phosphorus.  5.0  MANAGEMENT OF SITES DOMINATED BY ERICACEOUS SPECIES Following clearcutting or burning without prompt  regeneration of conifers,  it is common for old growth forests  with ericaceous plants already in the understorey, to become heath plant dominated cutovers.  Some examples are:  1)  Kalmia  barrens in Newfoundland following cutting of black spruce; 2) Vaccinium, Kalmia, and Ledum heaths in Nova Scotia or in the boreal forests following cutting of jack pine or black spruce on low productivity soils, and 3)  Gaultheria dominated cutovers  following clearcutting of old growth western red cedar forests on coastal British Columbia. Once established it is very difficult to eradicate the heath vegetation. use.  Herbicides are not usually effective or licensed for  Planting trees into dense heath cover is not feasible.  Burning usually stimulates further heath plant sprouting and renews their vigour.  In some cases the dominance of heath plants  can be very long term and represent a permanent exclusion of forest cover,  as seen in British heathiands and in Newfoundland 31  and Nova Scotia.  For boreal and westcoast heathlands, evidence  suggests slow invasion by trees and eventual forest reestablishment. Evidence from coastal western hemlock forests in Alaska suggests that periodic natural windthrow which uproots old trees and buries the humus layer, soil fertility (Bowers,  is an important factor in maintaining  1987).  In some British heathlands where  pure Sitka spruce stands suffer growth check and nitrogen shortages, mixed spruce and pine or spruce and larch stands show vigorous growth and no shortage of nitrogen (Malcolm,  1975).  To  date, there is no North American evidence for this “nurse crop” or mixed species effect. Reforestation of heathiands usually requires ploughing or use of backhoes to physically turn over or rip out the heath plants, usually followed by nitrogen and phosphorus fertilization prescribed on a site specific basis. chiorotic,  Current work with  slow growing Sitka spruce, western  hemlock and  western red cedar regeneration on Gaultheria dominated cutovers indicates that nitrogen and phosphorus provide an immediate but temporary release of growth check, whether applied at time of planting or later. European experience with attempts to reforest established heathiands indicate it is difficult, compared to preventative measures.  slow and expensive as Therefore, the most approp  riate actions are those designed to avoid establishment of heath plants such as prevention of fires on naturally regenerated 32  cutovers; seed bed preparation for prompt natural regeneration; and rapid planting,  supplemented where necessary by fertiliz  ation.  6.0  SUMMARY  From the review of the literature,  it is evident that the  problems of chiorosis and stagnation of conifer plantations associated with ericaceous plants, throughout the northern hemisphere.  such as salal, are similar The presence of ericaceous  plants can greatly affect forest productivity through a number of complex interacting mechanisms. Ericaceous plants have been found to be associated with organic acids such as phenolic acids, polyphenolics and fatty acids in soils which can be phytotoxic to plants, probably through their effects on root membrane permeabilities or mycorrhizal infections,  leading to a failure of cells to maintain  adequate mineral nutrition,  and the inefficiency of the energy  systems of respiration and photosynthesis.  The phenolic  compounds can polymerize and chelate with iron and aluminum in soils forming organo—metallic complexes which migrate and precipitate in lower mineral horizons leading to podsolization and possibly the formation of iron pans, thereby impeding drainage and rooting. The polyphenolics can also form tannin—like compounds with proteins and enzymes resulting in decreased decomposition and mineralization of organic material and the accumulation of raw humus.  The fatty acids are reduced by j3—  33  oxidation into octanoic, nonanoic and decanoic acids, which can be both phytotoxic and fungitoxic.  The overall result is a  decrease in the productivity of forests. Land management practices which allow the establishment of heathlands, results in expensive site specific efforts at rehabilitation, which are rarely fully effective.  Therefore, the  most appropriate actions are those designed to avoid establishment of heath plants such as prevention of fires on naturally regenerated cutovers, seed bed preparation for prompt natural regeneration, and rapid planting, supplemented where necessary by fertilization. Based on this literature review, it is evident that salal may be using allelopathic mechanisms such as the production of phenolic acids, polyphenolics such as tannins, or fatty acids which reduces tree growth and leads to an overall decrease in forest productivity.  This hypothesis is investigated in the  following chapters.  34  Chapter III  Soil Classification and Characterization of CH and HA Sites  1.0  INTRODUCTION The growth—check of conifer regeneration on CH but not HA  phases of the Thwia plicata shallon  —  -  Tsuga heterophylla  Rhytidiadeiphus loreus or “salal  —  -  Gaultheria  moss” ecosystem is  thought to be related to the reinvasion of the cutover by salal. However,  site conditions which encourage the growth of salal on  CH but not HA may in itself contribute to the differences in stand productivity.  The objectives of this study were to  document some of the differences in physical and chemical properties of mineral soil and forest floor humus horizons and to see if these properties differed between the CH and HA phases. It was anticipated that this could identify the factors responsible for the poor growth performance of conifer regeneration on the CH phase relative to the HA phase.  35  2.0  METHODS Soil and Vegetation Description and Collection  2.1.  Five sites each of the CH and HA phases were located near Port McNeill on Western Forest Products Tree Farm License 6.  The  sites were located such that an HA sites was adjacent to a CH site and were thus paired.  The sites were numbered such that odd  numbers were CII sites and even numbers were HA sites, thus paired sites would be numbered 1 and 2,  3 and 4,  et cetera.  Preliminary investigations of the forest floor horizons on CII and HA sites indicated that the organic materials were diverse in origin and state of decomposition which could cause inherent differences in chemical composition, biological activity and physical properties of the various forest floor horizons. Traditional methods of forest floor sampling involving the use of bulk samples could consequently cause results to be misleading or insignificant, unless prohibitively large numbers of samples were collected.  It was therefore decided to work within defined humus  horizons which would provide a more homogenous sample base than by random bulk sampling, and would also allow more effective communication with other investigators working on similar materials. Forest floor horizons were initially examined along two soil transects through typical CII (Rupert 206) sites.  and HA (Rupert 400)  The transects were run 15 m in a direction in which there  were no obvious changes in elevation.  Salal and other brush was  removed and the transect dug to a width of about 1 m and a depth 36  of about 1 to 2 m.  The forest floor and mineral soil horizons  were examined macroscopically for colour and texture, then representative samples of forest floor were returned to the laboratory for further analysis.  Air dry samples were again  examined for colour and texture, then oven dried and ashed. Obvious similarities and differences which could be used for quick and easy field identification were felt to be important to the classification.  The publication by Green  (1991)  on  humus form classification indicated ways of reporting more informative horizon designations and the approach was adopted. Once the horizon classification was determined,  samples were  collected for further analysis. Within each of the five previously chosen CH and HA stands, a 30 m transect was run through an area that appeared homogeneous in terms of topography and vegetation. plots were located 3 m apart,  Along this transect,  10  and soil pits were dug through the  forest floor and mineral soil to a depth where roots were absent, usually to a root restricting layer.  Vegetation within a 2 m  radius was noted (as described in the previous section).  Forest  floor and mineral soil horizons were classified and measured for depth, with estimates made of abundance and size of roots and the coarse fragment content of the mineral soil at depths of 0 to 10 cm and 10 to 20 cm. From each site, one or two samples best representing each humus type were collected in plastic bags and kept in a refrigerated truck until transported back to the laboratory where  37  they were stored at 4°C until processed.  Each sample was sieved  at field moisture through first an 80 mm sieve to remove large roots and wood pieces, then through a 40 mm sieve to break up small pieces of wood and remove fine roots.  Samples were halved,  with one half frozen at -30°C for phenolic acid determination, and the other half air dried, ground to pass a 0.4 mm screen and stored at room temperature in sealed plastic containers.  Laboratory Methods  2.2  2.2.1  Nutrient analysis  The nutrient analysis work was done at the U.B.C. Department of Soil Science in the Soil Chemistry Laboratory unless otherwise noted.  All nutrient concentrations are expressed on an oven—dry,  ash—free basis.  Soil moisture content was measured as the  difference between field moist and oven-dry weight after drying overnight at 105°C.  Ash content was measured on oven—dried soil  in a Thermolyne Furnatrol I muffle furnace.  The temperature was  set at 200°C for one hour, then raised to 450°C for three hours. Total carbon was measured by a Leco Induction Furnace (model 521)  and Leco Carbon Analyzer (model 572-200).  Total nitrogen  was determined by a semimicro—Kjeldahl procedure (Bremner and Mulvaney,  1982).  Total sulphur was determined with a Fisher High Temperature Furnace and Sulphur Analyzer (Models 472 and 475).  Available  sulphate S was estimated colorimetrically after HI—reduction of a  38  2 extract, CaCl  as described by Kowalenko and Lowe  (1972).  Mineralizable and available N were analyzed by Pacific Soils Analysis Incorporated using an anaerobic incubation at 30°C for 14 days  (Waring and Bremner,  1964),  then measuring the NH -N on 4  an autoanalyzer. Available P was analyzed at Pacific Soils Analysis Incorporated using the P extraction method of Mehlich (1978), as described by Lavkulich (1982). The exchangeable cations calcium (Ca), magnesium (Mg), potassium (K)  and manganese were analyzed by Pacific Soils  Analysis Incorporated using 1 M ammonium acetate extraction at pH 7 followed by atomic absorption spectrometry (Lavkulich,  2.2.2  1982).  Polysaccharides and cellulose  Total and labile polysaccharides were estimated using the phenol—sulphuric acid procedure of Dubois following acid hydrolysis.  1. (1956),  For labile polysaccharides,  hydrolysis was carried out by autoclaving for 1 hour at 15 PSI (103 KPa).  For total polysaccharides, hydrolysis involved cold  treatment with 72% 4 S0 which was subsequently diluted to 0.5 M 2 H , and then autoclaved as for labile polysaccharides Sowden,  1962; Cheshire,  1979).  (Ivarson and  Cellulose was calculated as the  difference between the total and labile polysaccharides.  2.2.3  Lipids  Lipids were measured at the U.B.C. 39  Soil Chemistry Laboratory  by shaking 5 g of sample with 75 ml of 1:1 ethanol-benzene for 2 hours and suction filtering. tared 250 ml beaker, weighed (Lowe,  The leachate was transferred to a  evaporated in a fume hood, and the residue  1974).  2.2.4  Bound phenolic acids  Bound phenolic acids were measured at the U.B.C. Chemistry Laboratory using an alkaline hydrolysis. soils were shaken overnight in 1 N NaOH.  Soil  Air-dried  The extract was  filtered and then acidified to pH 2 to precipitate humic acids and to fully protonate the phenolic acids.  The protonated  phenolic acids were then extracted in diethyl ether, which was allowed to evaporate, and the residue taken up in 3 ml of methanol for HPLC analysis.  Prior to injection into the HPLC,  samples were filtered through a 0.2 micrometer Prep-Disc Filter (Biorad).  Chromatography was done based on the procedure of  Drijber and Lowe  (1991)  using an acetic acid/acetonitrile  gradient and chromatographic conditions as follows:  Time  0 11 16 30 33 35 40 43  % 1% Acetic Acid 92 92 86 86 40 40 92 92  % Acetonitrile 8 8 14 14 60 60 8 8  40  Flow = 1.5 mi/mm Wavelength = 280 nm Temperature = Room temperature Attenuation = 16 (bound phenolics) Chart speed = 0.5 cm/mm Detector Sensitivity = 0.01 Standard = 30 ppm (bound phenolics) Peak Threshold = 22 Peak Width = 6 Column = ODS Spheri-5 25 cm (Brownlee) Guard Pak = p Bondapak C18 Inserts (Waters) Solvents = Acetic acid Aristar (BDH) Acetonitrile Omnisolv (BDH) Standards = Protocatechuic, p-Hydroxybenzoic, Vanillic, Trans—p—coumaric, Ferulic (Sigma)  2.2.5  Syringic,  Statistical analysis  All statistical analyses were done using SYSTAT System for Statistics  (Wilkinson,  1990).  homogeneity of variances, necessary.  and a log transformation done when  Statistical tests involved analysis of variance,  Tukey’s HSD test,  3.0  Bartlett’s test was run to ensure  covariance analysis and discriminant analysis.  RESULTS AND DISCUSSION  3.1  Site Description Sample sites are described in Table 3.1.  The elevations  of the site were similar, between 90 and 110 metres.  The HA  sites tended to be on ridgetops with 0 percent slope and aspect, while the CH sites were immediately adjacent on lower slopes, ranging from 11 to 20% slopes.  The CH site therefore, tended to  be wetter than the HA. The site characteristics were taken from inventory maps belonging to Western Forest Products.  The CH stands were  characteristically old, with estimates as great as 1000 years 41  SCHIRP  Rupert 400  Rupert 400  Rupert 206  Rupert 220  Misty 100  Misty 100  Misty 200  Misty 200  2  3  4  5  6  7  8  9  10  HB  CH  HB  CH  BH  C(H)  HB  C(H)  BH  C(H)  Forest Type  90  85  91  88  107  98  0  18  0  11  0  0  4  20  67 85  8  11  (%)  Slope  98  85  Elev. (ft)  350  330  45  20  45  0  Aspect (deg)  Dense  30.1-40  141+  141+  101—120  141+  81-100  40.1—50  40.1—50  30.1—40  40.1—50  30.1—40  Normal  Medium  Medium  Medium  Dense Normal  Medium  Normal  Medium  Dense  30.1—40  141+  Good Medium  Dense  30.1-40  82  Medium  Medium  Medium  Site Class  Normal  Normal  40.1—50  141+  82  Normal  40.1-50  141+  Density  Height (m)  Age (yrs)  42  Indicates the relative proportion of species within the stand: western red cedar (C), western hemlock (H) and amabalis fir (B).  SCHIRP  1  *  Location  Site Location and Characteristics  Site#  Table 3.1:  since last disturbance (Lewis,  1982).  Dominant tree heights were  above 40 in with the exception of stand 5 were younger (80 to 120 years old)  (30 m).  and denser.  The HA stands Site class was  medium for all stands except stand 4, which was rated as good. According to the Western Forest Products Inventory map, three of the CII stands had a greater proportion of western red cedar than western hemlock, and the other two had equal amounts of each. The HA stands tended to have equal amounts of western hemlock and amabilis fir, with two stands having a predominance of amabilis fir, and the other three of western hemlock.  All sites were  characterized as having moderately well to imperfectly drained Duric Humo—ferric Podzols arising from a sandy—loam glacial till with a blanket and rolling surface expression. Differences in vegetation between CII and HA sites were obvious, both in the field and statistically.  The CH stands were  more open and had abundant understorey vegetation, particularly salal  (occurring on 98% of the plots at a surface area of 66%),  while the HA stands were dense with very little understorey vegetation (Table 3.2). Howell and 31.  Vaccinium pp.  (including 31. alaskaense  ovalifolium Smith) were present with similar  frequency and abundance on both sites, as were the mosses Rhytidiadeiphus loreus (Sull.)  Ochyra.  (Hedw.) Warnst and Kindbergia oregana  In addition to salal, the only other constant  vegetation on the CII site was deer fern (Blechnum spicant (L) With)  and the moss Hylocomium splendens  (Hedw.)  B.S.G.  The HA,  because of its dense stand structure had sparse shrub cover 43  Table 3.2: vegetation (% presence and % cover on those plots where present) by stand number.  Stand Number and Phase CH 1  3  HA  5  7  9  4  2  6  8  10  Species Gaultheria shallon  100 100 100 100 90 68 19 95 3035  50 3  0 0  0 0  10 1  20 1  Vaccinium pp.  60 4  70 5  60 3  100 40 13  50 1  70 2  60 3  60 3  60 11  0 0  10 3  10 2  40 2  0 0  0 0  0 0  0 0  0 0  0 0  Rubus spectabalis  50 10  0 0  20 5  10 2  0 0  0 0  0 0  0 0  0 0  0 0  Blechnum spicant  80 13  70 8  30 17  10 1  30 2  30 11  20 2  20 3  10 2  0 0  Cornus canadensis  30 1  40 2  40 5  10 1  10 1  0 0  0 0  0 0  0 0  0 0  0 0  10 1  0 0  30 1  0 0  0 0  0 0  0 0  0 0  0 0  Lysichiton americanum 10 2  30 2  0 0  0 0  0 0  0 0  0 0  0 0  0 0  0 0  Montia siberica  10 1  0 0  0 0  10 1  0 0  0 0  0 0  0 0  0 0  Hylocomium splendens  100 100 100 100 90 46 30 25 54 42  70 33  40 35  50 1  Kindberctia orecrana  100 32  Menziesia ferruginea  Tiarella trifoliata  0 0  Rhytidiadeiphus loreus 30 23  60 19  60 19  80 50 31 66  50 10  100 51  60 13  60 40  80 50 15 17  90 33  90 21  44  100 100 23 18  50 100 100 7 30 23 10 10  70 6  60 28  including salal, Vaccinium pp. and occasional Rubus spectabilis Pursh.  3.2  Humus Classification and Variability 3.2.1  Hi.unus horizons  In the context of this paper, the term “humus” is used to describe any of the organic horizons of the forest floor.  The  forest floor was divided into master horizons based on the degree of decomposition, horizons  as described by Green  .  .,  (1991).  (L), which were generally very thin (< 1 cm)  Litter  and  consisting of the freshly fallen debris of the surrounding vegetation, were removed prior to sample collection of underlying horizons. The F and H master horizons were subdivided into 2 broad categories based on their woody or non—woody nature. wood were given the suffix “w”. Fw,  Those with  The woody horizons included an  in which the woody structure held when rubbed between the  fingers; an Hrw (residuic),  in which the woody structure failed  when rubbed between two fingers,  but consisting of greater than  20% woody materials; and an Hw consisting of less than 20% woody material.  Simple field tests that further distinguished the two  humic horizons include the appearance of dark coloured, greasy materials that rubbed out on fingers for Hw but not for Hrw; the reddish colour of Hrw versus the brownish red colour of Hw; and the more massive and compact structure of Hw (Table 3.3). The non—woody horizons consisted of 3 types. 45  A matted Fm  Table 3.3:  Physical Descriptions of Organic Soil Horizons on CH and HA Sites.  Horizon Composition  Colour  Structure  Rootinci  Fm  >60% plant <20% amorphous >20% fungi  1OR 3-4/4-6 2.5YR 3/4—6 5YR 2.5-3  compact, matted  abundant fine to coarse  Fw  >90% wood  1OR 3/4 2.5YR 3/4-6 5YR 3/4—6  woody structure holds  few  Hrw  <80% wood >20% amorphous  1OR 2.5—3 2.5YR 1—2 5YR 1—2  woody structure fails  few  Hw  <20% wood >80% amorphous  1OR 2.5—3 2.5YR 1-2 5YR 1-2  crumbly greasy  plentiful to abundant  Hh  no wood >80% amorphous  1OR 2.5-3 2.5YR 1-2 5YR 1-2  massive, blocky greasy  plentiful to abundant  Hhi  >95% amorphous  5YR 2.5-5 7.5 YR 0-1  massive, very few very greasy blocky to fine granular  46  (mycogenous) horizon,  containing abundant fungal hyphae and plant  roots; a well decomposed Hh  (humified) horizon, which was greater  than 80% amorphous, with a massive structure, greasy texture and a dark colour; and a Hhi, a very massive, very greasy, black horizon, greater than 95% amorphous, containing intermixed mineral particles (17—35% organic carbon mass),  found immediately  above the mineral soil.  3.2.2  HUmUS profiles  Humus profiles along the two trenches from the CH and HA sites were classified according to Green  .j,.  (1991)  illustrated in Figures 3.1 for CH and 3.2 for HA.  and are  Estimating the  proportions of various humus forms was done by simply dividing the length of humus type along each trench by the total length of trench; results are shown in Table 3.4. The HA trench consisted of 37% Hemimors Velohemimors),  22% Huntimors  (specifically  (8% Orthihumimors and 14%  Melahumimors), and 41% Lignomors (26% Hemilignomors and 14% Orthilignomors).  The presence of Hemimors,  in which the Fm  horizon comprises greater than 50% of the combined thickness of the F and H horizons,  occurs over recently windthrown mixtures of  organic and mineral soil.  The Lignomors consist of greater than  35% by volume of decaying wood, and are indicative of a windthrow event.  The Humimors have a well developed H horizon reflecting  maturity and a relative lack of disturbance. The CH trench has a greater diversity of humus forms 47  i  6  Figure 3.la.  ORTHILIGNOMOR  5  I I I  CH Trench Profile: O-6m  ORThOHUMIMOR  3  48  CH trench profile from 0 to 6 m.  Litter  4  I  Hw  Hrw  I  Hh  Fm Fw  LEGEND  ORTHILIGNOMOR  Non-woody  1  Woody  MELAH[. IORTHILIGNOM0RMjMOR,  2  0  Bfg  Bf  Figure 3.lb.  I  11  I  9  49  CH trench profile from 6 to 11 m.  UGNOHUMIMOR  10  MELAHUMIM0P  8  Litter  LIGNOHUMIMOR  Woody  7  Hw  Hrw  Fw  ] Hhi  Fm  Non-woody  LEGEND  MELAHUMIMOR  6  E  a  I’)  16  Figure 3.lc.  14  ORTHIHYDROMOR  13  50  CII trench profile from 11 to 16 m.  LIGNOHYDROMOR  15  I  Bfh  HEMIIIYDROMOR  12  Hw  Hhi  Fm Fw Hrw  Non-woody  Woody  LEGEND  LIGNOHUMMOR  11  E a  If)  Figure 3.2a.  4  Aheb  Bfh2 Bfh3 Bfh4  2  —  51  HA trench profile from 0 to 4 m.  Bhfu  S  Litter  VELOHEMIMOR  3  Bf  ORTHIHUMIMOR  Woody Fw  E 0  to  Fm  Non-woody  LEG END  HEMILIGNOMOR  0  -S  -S  -S  -S  -S  Ah  9  5-  Bf  Figure 3.2b.  -S  7  Litter  52  HA trench profile from 4 to 9 in.  HEMILIGNOMOR  8 6 VELOHEMIMOR  5  Woody  Bhfu  Hrw  Fw  Hh  Fm  Non-woody  LEG END  4  E 0  I’)  14  Figure 3.2c.  Litter  12  Bf  MELAHUMIMOR  11  53  HA trench profile from 9 to 14 in.  ORTHILIGNOMOR  13  Fw  Hw  Fm  Non-woody  LEG END  HEMILIGNOMOR  Hrw  Woody  10 9  E  d  LO  Table 3.4:  Proportion of humus forms on CH and HA trench profiles.  ORDER  SITE Subgroup  CH  HA  HEMIMOR Velohemimor Melahemimor  0 2.3  37.4 0  HUMIMOR Orthihumimor Melahumimor Lignohumimor  18.8 9.2 28.7  7.6 14.2 0  LIGNOMOR Orthilignomor Hemilignomor  17.3 0  14.4 26.4  HYDROMOR Orthihydromor Hemihydromor Lignohydromor  7.6 4.5 11.7  0 0 0  54  including 2% Hemimors  (Melahemimors),  57% Humimors (19%  Orthihumimors, 9% Melahumimors, and 29% Lignohumimors), Lignomors (Orthilignomors), and 24% Hydromors 7% Orthihydromors and 12% Lignohydromors).  17%  (5% Hemihydromors,  In comparison to the  HA trench, the CH has had few recent disturbances,  as indicated  by the relatively large proportion of Humimors and small proportion of Hemimors and Lignomors.  The Hydromors, which were  not found in the HA trench, develop under the influence of fluctuating,stagnant water that is generally less than 50 cm below the ground surface,  in this case over Gleysolic soils.  A  closer examination indicated that the gleyed soil occurred over a highly cemented mineral horizon, which seemed to impede drainage.  3.2.3  Soil variability  To determine the variability between CH and HA sites, the ten plots from each of the five CH or HA stands were combined to give a total of 50 plots for each of the CH and HA sites. Characteristics were averaged based on occurrence. The soils on the HA sites were found to be drier and less compacted than on the CH.  For example,  64% of CH plots had  standing water occurring at an average depth of 23 cm, while for the HA sites,  only 16% of the plots had standing water,  at an average depth of 24 cm (Table 3.5).  occurring  Root restricting  layers, such as cemented or compacted horizons or standing water, occurred in 98% of the CH plots, but in only 70% of the HA plots. The mean rooting depth in the CII was thus significantly shallower 55  Table 3.5:  Site property values including mean, standard deviation and occurrence (% of plots) on the CH and HA sites.  Site Type Property  CH  HA  Seepage Depth  (cm)  22.6 (15.1) 64  24.0 (4.4) 16  Rooting Depth  (cm)  21.4 (19.6) 100  28.8 (18.0) 100  Root Restricting Layer Depth (cm)  21.0 (16.5) 98  21.3 (14.7) 70  % Coarse Fragment Content (0—10cm)  19 (14)  17 (17)  % Coarse Fragment Content (10—20cm)  51 (25)  32 (25)  Total Forest Floor Depth (cm)  23.0 (13.6) 100  28.8 (16.8) 100  Follisol Depth  48.3 (9.4) 7  56.5 (10.9) 11  56  (21 cm)  than the HA (28 cm).  Also, the coarse fragment content  of the CH was significantly higher in the 10 to 20 cm depth than the HA. The overall depth of humus was greater for the HA (29 cm)  than  the CH (23 cm), and this was because the HA had a greater quantity of decaying wood than CH (Table 3.6). the findings of Lewis horizons  (Fw and Hrw),  (1982).  This contradicts  The least decomposed woody  occurred in 68% of the HA plots, with a  mean depth of 20 cm, while they occurred in 38% of the CII plots with a mean depth of 19 cm. 56% of the HA plots (15 cm depth).  The well decomposed 11w was found in  (16 cm depth), but only 38% of the CH plots  The rionwoody Fm and Hh were found in virtually  all of the CH and HA plots, but the depth on the HA (6 cm) was significantly less than on the CH (9 cm).  The well humified Hhi  occurred on 32% and 28% of the CH and HA plots respectively, but the depth on the CH was significantly greater than the HA (5.2 cm and 2.5 cm respectively). than 40 cm)  Folisols  (forest floor depths greater  occurred on 22% of the HA plots  14% of the CH plots  (48 cm depth).  (57 cm depth),  and on  Overall, the HA tended to  have deeper forest floors with significantly more woody huiuas than the CH, which had significantly more non-woody humus.  The  high occurrence of rotting wood on the HA reflects the relatively recent catastrophic windthrow event of 1908.  The woody horizons  found on the CH reflect single tree blowdowns. The mineral soil horizons were examined superficially and horizons designated by experience using colour and texture; no 57  Table 3.6:  Forest floor horizon depths including mean, deviation and occurrence (%) by site.  Horizon  CH  HA  Fm—Hh  8.7 (4.5) 98  6.3 (3.3) 92  Hhi  5.2 (3.1) 32  2.5 (1.3) 28  Fw—Hrw  18.7 (15.2) 38  19.9 (14.2) 68  Hw  15.0 (8.8) 38  15.6 (9.6) 56  58  standard  chemical analyses were done. were made  (Table 3.7).  Several interesting observations  The CH plots tended to have deeper and  more consistent occurrence of A horizons Ahe), probably from lack of disturbance.  (including Ah, Ae and The HA plots had a  greater occurrence of organic enrichment in the B horizons, probably burial of organic horizons during windthrow.  The CH had  a much higher occurrence of gleyed horizons than the HA.  The HA  tended to have much more friable mineral soils, while the CH was more compact. These general observations along with the more empirical information seems to indicate that the windthrow process is very important to the rejuvenation and higher productivity of HA sites.  The windthrow process not only breaks up pans and other  cemented horizons,  but also mixes mineral soil with humus and  woody materials which aerates the soil,  increases the friability,  and encourages deeper rooting by enriching the mineral soil fertility.  In contrast, the CH site, which have had very little  disturbance, have almost continuous root restricting layers and a high occurrence of standing water over some portion of the year. The formation of Hydromors, occurring under periodic anaerobic conditions,  is associated with accumulation of organic compounds  during the hydromorphic period.  When the profile dries out,  these organic acids migrate down and behave as active agents of podzolisation and complex formation (Bloomfield, without a major disturbance event,  1975).  Thus,  such as windthrow, CH sites  would tend to degrade further with a greater occurrence and 59  Table 3.7:  Mineral soil horizon depth (cm), standard deviation, and occurrence (% of plots) by site type. (+ indicates greater than).  Horizon  Site Type CH  HA  Ah  4.5 (2.7) 17  2.9 (1.3) 19  Ahe  6.7 (3.8) 37  4.2 (2.3) 36  Ae  2.6 (1.5) 9  0  Bh  9.5 (8.1) 4  7.8 (4.8) 9  Bfh  14.3 (13.5) 28  12.1 (11.0) 36  Bf  11+  15+  18  20  Bfgh  14.1 (8.4) 11  14.0 (2.8) 2  Bfg  14+  11+  11  2  15+  14+  8  8  Bg  60  build—up of Hhi humus horizons, cemented horizons,  and standing  water.  3.3  Nutrient Concentrations of Humus Horizons 3.3.1  Nutrient concentrations of woody horizons  Woody horizons develop from logs decomposing in place on the forest floor, with a gradual build—up of forest floor over them. There is virtually no mixing of woody material with non-woody material until they approach a more decomposed stage, when they begin to turn dark and lose the characteristic red colour.  The  Fw and Hrw therefore have very low ash contents and very low quantities of total and available nutrients  (Table 3.8).  As  decomposition proceeds, there tends to be an accumulation of nutrients, presumably from decomposer organisms, and mixing with non—woody and inorganic materials,  so that the ash content  increases significantly, as does total N, total S, mineralizable N,  and available N.  As expected, C/N ratios decreased  significantly with decomposition within the woody humus types, decreasing by almost half from Fw to Hrw, and again from Hrw to 11w.  This occurs because of a reduction in total C and a  simultaneous accumulation of total N, which almost doubles from Fw to Hrw, and again from Hrw to Hw. with decomposition is well documented.  This decrease in C/N ratio According to Stevenson  (1981), the decay of organic residues by soil organisms leads to the incorporation of part of the C into microbial tissue, with the remainder being liberated as CO . 2  61  At the same time, organic  Table 3.8:  Mean nutrient concentrations and standard deviation by horizon on oven—dry and ash-free basis. Similar letters indicate no significant difference between all horizons. Woody  Non-Woody  Fw  Hrw  Hw  Fm  Hh  pH 0) 2 (H  3.67ab (.07)  3.61a (0.11)  3.56a (0.11)  3.92ab (0.51)  3.59a (0.15)  4.16b (0.42)  ash  0.9a (0.3)  1.3a (0.7)  2.9b (1.0)  4.8b (3.0)  5.7b (4.1)  34.Oc (13.2)  56.5c (2.2)  56.3bc (2.4)  54.8bc (0.7)  50.8bc (0.9)  53.2bc (1.1)  60.3a (19.2)  0.286a (0.130)  0.479b (0.171)  0.876c (0.217)  1.115c (0.202)  1.lOlc 1.752d (0.142)(0.324)  665a (157)  920b (180)  1585c (254)  1649c (256)  1766c (140)  3132d (910)  C/N  226d (77)  127c (32)  66b (17)  47ab (10)  49b (8)  35a (9)  Avail. N (ppm)  23a (5)  29a (12)  34a (11)  61c (13)  61c (31)  61c (19)  Avail. (ppm)  P  8a (3)  9a (2)  9a (4)  33c (15)  18b (9)  9ab (2)  Avail. (ppm)  S  23a (10)  25a (7)  27a (5)  64b (26)  Sob (16)  44b (23)  Exch. Ca* (me/bog)  6a (2)  8a (6)  8a (5)  13a (9)  13a (7)  ha (11)  Exch. Mg* (me/bOg)  3a (1.2)  6ab (3.3)  7ab (3.5)  4a (0.7)  9b (2.9)  6ab (5.5)  Exch. K* (me/bOg)  O.4a (0.2)  0.6a (0.4)  0.6a (0.2)  2.3c (0.9)  1.2b (0.4)  O.7ab (0.3)  Extr. Mn (ppm)  15a (11.7)  22a (43.8)  4a (4.9)  207b (201)  21a (17.0)  14a (18.9)  (%) C*  (%) N  (%) S*  (ppm)  *  Variances equal after log transformation.  62  Hhi  N is converted to available N (NH 3 and NO -) which soil organisms 3 utilize for the synthesis of new cells. fixed into humic substances.  Also,  available N can be  The result is a gradual  transformation of plant material into stable organic matter with a fairly consistent C/N ratio. Similarly, total S accumulates significantly with decomposition, (Fw)  from an average of 665 ppm in the least decomposed  to 1585 ppm in the most decomposed (Hw).  Total N and S  concentration are not significantly different between the Hw horizon and the Hh horizon,  indicating that N and S  concentrations in woody materials accumulate as decomposition proceeds,  towards that of well decomposed non—woody materials.  The available nutrients N, P and 5, were not significantly different between woody horizons. nutrients,  Similarly, the inorganic  Ca, Mg and K were not significantly different, but  there appeared to be a trend towards increasing concentration with decompostion.  3.3.2  Manganese was extremely variable.  Nutrient concentration of non-woody horizons  The non—woody horizons are formed from the input of litter to the forest floor,  including leaves, roots and fruits of the  plants growing in the immediate area.  Nutrients are more  concentrated in litter material than in wood, and the horizons derived from litter therefore, have significantly greater total N, total S,  and available N, P and S than the woody horizons.  The relationships between the non—woody horizons are more complicated than between woody horizons.  For example, the ash  content of the Hhi is very much higher than that of either the Fm 63  or the Hd,  presumably because of the long residence time and the  close proximity of the Hhi horizon to the mineral soil. Therefore,  on an oven—dry ash—free basis, the concentrations of  C, N and S appear to be significantly greater than that of the Fm and Hd (Table 3.8), whereas,  on an oven—dry basis alone, the  concentrations of C are significantly lower, and that of N and S not significantly different (not shown).  Similarly, the  available nutrients N, P,and S are not significantly different on an ash—free basis, but are significantly lower on an ash basis (not shown). There were no significant differences between the Fm and Hd horizons for total C, N,  S,  and available N and S, but available  P was significantly lower for the more decomposed Hd.  Although  not significant, there appeared to be a trend towards decreasing concentrations of available N, P and S from Fm to Hd to Hhi.  A  larger number of samples would likely improve the significance, consistent with natural trends in decomposition towards more resistant,  less available nutrient forms.  For the inorganic nutrients, the only significant differences in the non—woody horizons were that Mg was significantly higher in the Hd, K significantly lower for the Hhi,  and Mn significantly higher for the Fm. 3.3.3  Nutrient concentration differences between sites  The nutrient concentrations for each horizon by site are shown in Table 3.9 and Figures 3.3 to 3.8, Variance probabilities for horizon, interaction are shown in Table 3.10. 64  and the Analysis of  site and horizon X site As previously described,  significant differences between horizons were found for all the nutrients except Ca.  Generally, the non—woody horizons were  significantly higher in nutrients than the woody horizons. Significant interactions between site and horizon occurred for total N, total S and C/N ratio (Table 3.10).  The 11w horizon  had significantly greater total N and S and significantly lower C/N ratio on the HA site than the CH,  but there was no  significant difference between sites for the other horizons. The site effect and the horizon X site interactions were significant for the available nutrients N, P and S.  The Hh and  the 11w horizons from the HA site had significantly greater available N than the CH sites,  and the Hrw and the Hhi horizons  had greater available N, although not significantly so.  Neither  of the F horizons were significantly different in available N between sites,  indicating that mineralization tends to be lower  in more humified horizons on the CH site than on the HA. The HA site had significantly greater available P in the Hrw, Hw, and Hhi horizons than in similar horizons on the CH site.  There were no significant site differences for the woody  or non—woody F horizons,  or the Hh.  Again, this seems to  indicate that P is less available in more humified horizons of the CH than of the HA. process,  Turnover of organic P is a biological  and mineralization and immobilization are strongly  influenced by the physical and chemical properties of the soil. Differences therefore, must be a function of site. Finally, the CH site has significantly greater S availability in the Fm and Hrw horizons; greater, but not significantly greater, available S on the Fw and Hhi horizons; 65  01  (5)  ‘--‘9)  01—’. I.) HW O1.D 0101 01  9)  .P---’.H H—)  9)  01—. H HOl  5--’  H H4’01W 0101  9)  —H )0 010 014’  -.) HO 001  5—  .—. Cl)  01—. W HOl  ‘--‘9)  9)  9)  9)  W—-’. W 01W  ‘—-.01 .DH  ‘—‘9)  O.—. . H0 H  M—s. WOl  9)  01—. .1’.D01  -.3--. —.3 H01 01  01—. 01 H.t0 ‘—9) ‘—  9)  9)  010 ‘-‘  .—.I-WH 0IH  01—.H  s).P.  9)  —  01—.. H —JO I-J’.D  9)  ts) 01—) OW  Is  (5)  Z  C)  W—. H O• CI • HOl ‘.0--.) W 01—.. 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I-J. 0 I-’ )-b’.Q P) CD b  I  .  :i:  C  I  (-t-w z  C 0,  W’  CD  Prt  I  CD  0  z  II H) b H) CD CD .  1  0 CD  CD< P)C) C) I 0 CD I-’Z  I  bOCD CD I-’Z  rtCD r CD P)  CD ct 0 (1)0  -  I-’.  ct0  .L  CDtIO -.-  0010010010  01001  (1)  -n -D C 0(1)  C•)  0  CDb  I  C) CD  *  QrJ) I- i-a.  (I)  o  CD •  I-I.  I-’C•) —I-’ -.%  1  I C  I  0 D  C Co  I  C) 1 0  0  I-..  -I  3  I-i.  CO  I-,. C)P) Ct) CD I-’ (1)1-’  1  *  ‘-I  CD  Table 3.10:  ANOVA for Horizon and Site (Probability of Null Hypothesis) and Squared Multiple R.  Horizon  Site  Horizon*Site  2 R  N  .000  .048  0.890  S  .000  .081  0.900  C/N  .000  .035  0.884  AvN  .000  .000  .005  0.791  AvP  .000  .073  .044  0.765  AvS  .000  .002  .041  0.714  Ca  —  —  —  0.246  Mg  .002  K  .000  Mn  .000  TPSS  .000  .036  0.642  CELL  .000  .053  0.452  0.440 .000 —  .080 —  72  0.835 0.684  and no difference in available S on the Hw and Hh horizons (Figure 3.7). According to Stevenson (1982), the amount of S mineralized does not appear to be directly related to soil type, total concentrations of C, N, or S, or to C/SI N/S or C/N ratios, soil pH,  or mineralizable N, but is probably caused by the  chemical nature of the decomposing fraction and by those factors which influence the growth of microorganisms, such as temperature, moisture, pH,  and availability of food supply.  For inorganic nutrients, there were no significant effects of horizon,  site or horizon X site interactions for Ca (Table  3.10 and Figure 3.8).  Table 3.9 does show that Ca concentration  in the Fm horizon of the CH site was much higher than for the HA site,  and this is presumably because the litter of both western  red cedar  (Minore,  1983)  and salal  (Klinka,  1976)  tends to be  calcium—rich, but the large sample variation probably masks any significance. Similarly, the CH tended to have higher concentrations of Mg and Mn  (Figure 3.9),  but the differences were not significant,  again probably because of the high sample variation.  One  exception is that the Mn concentration in the CH was extremely and significantly higher for the Fm of the CH. pers.comm.) sites,  (1991,  found that Mn tends to be higher on nutrient—poor  and Klinka  (1976)  found the mean Mn concentration in salal  leaves to be over 2000 ppm.  1  1 Carter  It is unclear whether salal  Carter. Research Associate, R. Columbia, Vancouver, B.C. 73  University  of  British  ‘  rJ 1  I-.. II CD  I I-’. I-’.  rtb (J)CD wI-J I . 1  I  C  I  C (0  CD  I 0  0 C  II Ii 0 00  I  -‘  ZC) ct(D I  II CD I-• (DZ  0 (DO  frt,  ct  I-.. cttfl CD I-• tflrt CD  •  0 CD  I-1 1<  0 I-I.  *  Izj  I-,. -  ...h  h  -  h  II CD  C) 0)  m  I-J. 0 H, I- bi  :3-  (DNX 00  :3  cc2 0  CD  i  CD bQ ‘<CD b ) (Dflb ç1 I-’- I-  CD  C)  rfCD  CDCD CD 0  C.)  ‘—.  O’O  I_I  3  (noo < I-I.  I-I.  rtCD uI  I.<p) _  —  0) (Ji  o CD CD  o  -‘  rz  -  c,  c)  .o.cn.on CD  I  ‘1  C, 0  00  I-’. D  0  CD  ‘-JO)  z  * 1  CD 0) * CD  0)  m  -I.  0  I  :3.  C  ‘-I.  0.0 00)  C Co  CD  I 0  0  0  0)  0  O)U) cttn CD I-•  *  Ci)  CD  CD  -D 0 0  I  0  I.J. ‘.0  C  3  I-’• H ‘-I.  I  D  rtu  H 0  TJ  I-i. -  ‘I CD  -‘  h  DO. •000000  ‘.3 ‘.0  —  H) ti (DNX  ‘Ion  ‘.1  m  I -I  C)  I  0  I  0)  C C’)  CD U)9) O CD bQ  CD C) CD  CI)  tnoz  C)  ct -‘• I-’ rt(D CDCD CD 5 .—. 0) -  CD  < CD  ct(D U) CD S i  3  rn  I  .1-1  0; (1)10) Fs.) 1%) Cli 0(31 0 Cli 000000  CD U) O(D  00 • 0(310 000  ‘1  C)  ‘-I.  0 z  I-in  0  •P) S  CD  I -‘  m  -‘  CD *0)  .—‘  I-’. I-’ 00) 0)5 rtQ (DO) S CD U)  0)  I  C  0  C)  I CD  C CO  0 -‘ 0 (0 0)  I  0  *  3  Co  CD  (0 C’) 0  55 I-.. I-h  I  I-,. 0 OH) 0)  C)  CD  C) D  C)  D CD C’) CD  accumulates Mn and therefore leaves a high concentration in decomposing litter, or if Mn is a symptom of poor site conditions. The one inorganic nutrient which was significantly different by horizon and site was K.  Concentrations of K were  significantly higher in Fm and Hrw horizons from the CH site than from the HA, and higher,  but not significantly so,  horizons of the CH (Figure 3.8).  for all other  This high concentration may  reflect the presence of salal litter which has been shown to have relatively high concentrations  3.4  (6600 ppm)  of K (Klinka,  1976).  Organic Composition of Humus Horizons 3.4.1  Lipids  Lipids come from two basic sources,  from undecoinposed plant  materials and from the .bodies of dead and living microfaunal organisms.  Plant materials normally contain waxes which protect  the surface of leaves, trunks,  flowers and fruits.  particularly resistant to decomposition,  These are  and tend to accumulate  as other readily digestible carbon sources are utilized.  Not  surprisingly then, wood, which is basically lignin and cellulose, has a lower amount of lipids than non—woody horizons which are composed of many of these lipid-rich plant structures (Table 3.11 and Figure 3.10).  Microorganisms also synthesize lipids;  bacterial cells contain from 5 to 10% lipids, contain from 10 to 25%  (Stevenson,  1982).  fungi usually  As decomposition  proceeds, the bodies of dead organisms accumulate, so that the 77  Table 3.11: Organic composition of humus types including lipids, total polysaccharides, labile polysaccharides and cellulose by site-type based on 4 replicates (unless otherwise noted). Woody Fw Lipids  Hrw  Non-woody Hw  Fm  Hh  Hhi  (% oven-dry, ash—free soil)  CH  2.36 (1.47)  4.25 (1.21)  7.56 (1.29)  5.24 (0.42)  5.68 (1.14)  5.41 (2.11)  HA  1.93 (0.13)  2.22 (0.64)  5.04 (0.49)  4.86 (0.40)  4.82 (1.03)  2.62 (1.58)  Mean  2.15 (1.07)  3.23 (1.38)  6.30 (1.61)  5.05 (0.35)  5.39 (1.09)  4.21 (2.30)  Total Polysaccharides  (% oven—dry, ash—free soil)  CH  19.7 (14.9)  13.3 (1.9)  16.0 (3.5)  32.1 (2.2)  24.3 (4.8)  18.5 (8.1)  HA  11.1 (3.0)  12.4 (3.9)  14.8 (2.3)  26.6 (2.8)  21.6 (1.1)  15.8 (3.3)  Mean  15.8 (11.5)  12.5 (3.0)  15.1 (2.9)  28.1 (3.5)  22.1 (4.2)  17.2 (5.1)  Labile Polysaccharides  (% oven-dry, ash-free soil)  CH  12.9 (9.2)  10.4 (1.3)  13.2 (3.0)  26.2 (2.9)  20.3 (3.2)  15.3 (5.4)  HA  7.9 (1.7)  9.5 (2.0)  12.6 (1.5)  21.4 (1.3)  17.3 (0.3)  14.3 (3.1)  Mean  10.6 (7.0)  9.6 (1.6)  12.6 (2.3)  22.9 (3.3)  18.3 (3.2)  14.8 (4.8)  Cellulose  (% oven-dry, ash-free soil)  CH  6.8 (5.8)  2.9 (0.7)  2.9 (0.7)  5.9 (2.0)  4.0 (1.6)  3.3 (2.8)  HA  3.2 (1.4)  3.0 (2.0)  2.2 (1.1)  5.2 (1.5)  4.3 (0.7)  1.5 (0.8)  Mean  5.2 (4.6)  2.9 (1.5)  2.5 (0.9)  5.3 (1.6)  3.9 (1.3)  2.6 (1.8)  78  0  ITJ  I-’. II CD ‘1 0  I-&.  C  C)  I  I-’.  0  C)  III  CD  I V  c. Ci)  zri II  rt x  0 0  I  (0  0 N  0 (0  (0 I-s.  r1 CD  0 CD  z  1<  (0  T CD CD (0  0  I-I. I—. (0  content of lipids in more decomposed materials is higher. The lipid concentration of the humus types ranges between 1 and 9% which is in the reported range for soils means are shown in Table 3.11.  (Stevenson,l982);  There were no significant  differences between humus types.  However, two trends are clear.  First, the non-woody materials tend to have a higher lipid content than woody materials.  Second, there tends to be an  accumulation of lipids from the least decomposed to the most highly decomposed for both woody and non-woody types.  Both these  trends are expected. Differences between sites for each humus type are shown in Table 3.11 and Figure 3.10.  Again, differences are not  significant, but CH sites have consistently higher lipid contents and greater variability than HA sites.  There are several  possible reasons why lipid concentrations are higher on the CH: the concentration of lipids originating from salal or western red cedar litter may be higher than that of litter found on the HA; the wetter site conditions of the CH may inhibit the breakdown of lipids by microorganisms leading to accumulation; or to a larger quantity of lipids synthesized by microorganisms on the CH. intensive sampling might provide clues.  More  In any case, the higher  concentration of lipids in the CH would suggest that decomposition on these sites is slower, the HA.  or less complete than on  The higher concentrations of lipids on the CII suggests a  role for lipid oxidation products as allelochemicals, but this was not investigated. 80  3.4.2  Polysaccharides and cellulose  Polysaccharides are high molecular weight sugar compounds. They originate from plants, mainly cellulose and hemicellulose, and as products of microbial metabolism.  The stability of  polysaccharides in soils is caused by a combination of several factors which makes them resistant or inaccessible to microbial attack,  including structural complexity,  minerals or oxide surfaces,  adsorption on clay  formation of insoluble salts or  chelate complexes with polyvalent cations, and tanning by humic substances (Stevenson,  1982).  Total polysaccharides in woody horizons were not significantly different from each other, but were significantly lower than the non—woody Fm and Hh, which presumably contain abundant leaf and root residues  (Table 3.11 and Figure 3.11).  The well humified Hhi had significantly lower total polysaccharide concentration than the Hw.  The labile  polysaccharides did not have similar variances so could not be statistically analyzed,  but the trends appear to be similar to  that of the total polysaccharides (Table 3.11 and Figure 3.11). Cellulose, which was calculated as the difference between total and labile polysaccharides, accounted for only 2 to 5% of the total polysaccharides  (Table 3.11 and Figure 3.12).  There  were no significant differences in cellulose between the two F horizons,  Fm and Fw, and there was no difference between the two  most decomposed horizons, Hhi and Hw, but the difference between the most and least decomposed horizons were significant.  81  Horizon  LsJ  I•Tj  I-I. h  CD  0 •0  Cli 0  h  1%.)  1%)  Cs)  0  0  0  pio  p  001  0  0  Cs)  Cli 0  L)  •1  uO  I  C  3  C  Co I.z  NO O-l  I 0 N 0  z  I-’. II rt i-• CD * O <0 CD I.-.. -  p 0  !•  r.)  -  ç.n  P  0  0  0  00  n  p  C,)  p 0  I  0)  (OCD  CD  I-hO  -U  0  I-’. 1:r  C’) 0)  III.-, (D’< CDU2 0) (no  o  00 .—  -  ) -  =  CD  I-a.  D  CD  o  0 0  0. CD  CD  —  C)  0  •  —  r1  CD  0  -‘. -  ..  D  0 0  I.t,  a, G)  ‘4-I (0  >1  a) 0 *  a) U)  (I) 0  N  •d  0 (0  co  ‘4-I  0 0  4.)  x C  a) U  EN  0  C.) w  4.)  O  IJL 0  z  II  0  (0  •1  0  0 C 0  C)  a) (0  0.  I-I--..  4-  a)  U  II  rIr-I i1 rl  wo  I..  a) (N i-1  LL.  Cl  a) z •r-I  and site effects were significant (Table 3.10), but there was no significant interaction between horizon and site.  When total and  labile polysaccharides were compared by site within horizon, the CH was significantly higher for the Fm, and higher but not significantly so for all other horizons. was higher, but not significantly so,  Similarly, cellulose  for all horizons on the CH.  A greater sampling intensity to reduce overall variability would increase the significance.  The trends are in agreement with  earlier findings of lower nutrient availability and higher lipid contents on the CH,  and that decomposition seems to be slower or  less complete on the CH than on the HA.  3.4.3  Bound phenolic acids  The phenolic acids, p—hydroxybenzoic, vanillic,  protocatechuic,  and syringic acids (hydroxybenzoic acid derivatives),  and p—coumaric and ferulic acids  (cinnamic acid derivatives), are  commonly found in living plants and in soils.  The kind and  quantities of these phenolic acids in soil depends to a large degree on the origins of organic material.  Gymnosperm lignin  consists predominantly of coniferyl units with low amounts of p— coumaryl units and low or absent syringyl units; angiosperm dicotyledon lignin contains an equal amount of coniferyl and syringyl units with very low p—coumaryl units; and monocotyledons contain approximately equal amounts of all three units 1981).  (Crawford,  The p—coumaryl units can also be derived from suberin,  and to a lesser degree,  from cutin  84  (Kolattukudy,  1980).  Protocatechuic acid is a common intermediate product formed during catabolism of lignin (Kirk,  1984).  Thus in this study,  woody horizons can be expected to be high in vanillic acid, while the non—woody horizons, with a greater angiosperm contribution, would have higher syringic acid content.  Similarly, the litter-  derived Fm horizon would have higher coumaryl derived units than woody Fw horizon. As expected, a comparison of humus horizons  (Table 3.12)  indicates that woody horizons tended to be higher in vanillic acid while the non-woody horizons tended to be higher in protocatechuic, p—coumaric and ferulic acids.  The Fm horizon,  with the greatest proportion of angiosperm inputs, had the highest concentrations of protocatechuic,  syringic, p—coumaric,  and ferulic acids; the Fw, which is pure coniferous wood, had the highest concentration of vanillic acid; the Hhi, was found to be highest in p-hydroxybenzoic acid.  Interestingly, the Fm, which  is the most active horizons, was found to have the highest overall concentration of phenolic acids, while the Hhi, which would be expected to accumulate the mobile organic acids from overlying horizons, was found to have the second highest overall concentrations of phenolic acids. Changes with decomposition of woody humus were examined with no significant differences, but the following trends were found (Table 3.12).  Vanillic acid decreased with decomposition, which  was consistent with the reduction in total lignin content content  85  Table 3.12:  Concentration of bound phenolic acids (standard deviation) by humus horizon (micrograms per gram of oven—dried, ash—free soil).  ACID  HORIZON Woody  Proto— catechuic p—Hyroxy— benzoic Vanillic  Fw  Hrw  114a  120a  9Oab  361b  Non-woody  Hw  Fm  Hh  Hhi  121a  392b  342b  141a  52a  116b  108b  123b  248b  278b  279b  135a  161a  294b  Syringic  lBab  lOa  l7ab  35b  l6ab  26ab  p-Coumaric  3Oab  ha  l7ab  125c  83bc  75bc  Feruhic  23ab  h2a  l6ab  75c  52bc  71c  86  as discussed inprevious sections. (1986).  Kogel  A similar trend was found by  Syringic was found to initially decrease from Fw to  Hrw, but then increased from Hrw to Hw.  This may occur because  rooting density is greater in the more decomposed Hw, and could therefore be directly affecting the concentration of syringic acid. A comparison was made of phenolic acid concentrations between CII and HA (Table 3.13).  The Fw horizon, which had the  highest vanillic acid concentration was not significantly different between the two sites.  For the Fm horizon,  the CH site  was significantly lower in vanillic acid than the HA site, but significantly higher in syringic acid, which is consistent with the fact that the CH has higher angiosperm litter inputs.  There  were no significant differences between CH and HA sites in any of the humus horizons for protocatechuic,  ferulic or p—coumaric  acids. A correlation analysis was performed to examine the relationship between the concentration of bound phenolic acids and a) whether or not the horizon was woody, the plot covered by salal, or a)  b)  the proportion of  the proportion of the plot  covered by any other herbaceous or shrubby vegetation including Vaccinium spp., spicant  Cornus canadensis (bunchberry)  (deer fern).  and Blechnum  Vegetation data was taken from Table 3.2 in  the preliminary study which examined vegetational differences between CH and HA sites.  The results are shown in Table 3.14.  87  Table 3.13: Concentration of bound phenolic acids (standard deviation) by humus horizon and site (micrograms per gram of oven—dried, ash—free soil). ACID  SITE  Proto— catechuic  CH  p-Hyroxy-  Fin 483 (250)  Fw 113 (19)  Hrw 151 (54)  Hw 108 (39)  HA  302 (51)  116 (11)  90 (30)  135 (49)  CH  111 (44)  72 (28)  83 (24)  136 (44)  HA  105 (13)  109 (64)  22 (9)  96 (15)  CH  107 (13)  357 (45)  275 (48)  245 (80)  HA  162 (33)  365 (54)  281 (39)  312 (48)  CH  46 (14)  15 (4)  12 (3)  17 (6)  HA  24 (7)  20 (8)  7 (3)  17 (4)  CH  129 (96)  19 (19)  16 (10)  24 (16)  HA  121 (12)  40 (23)  6 (6)  10 (4)  CH  89 (21)  29 (18)  17 (13)  19 (5)  HA  62 (8)  17 (5)  7 (4)  14 (4)  benzoic  Vanillic  Syringic  p—Coumaric  Ferulic  HORIZON  88  Table 3.14:  Phenolic Acid  Pearson correlation matrix between bound phenolic acid concentration and abundance of salal or other shrubs, presence of wood, and site type (CH or HA).  Salal  Other Shrubs  Wood Presence  Type  Site  Protocatechuic  .278  .036  —.623  —.193  p—Hydroxybenzoic  .423  .486  —.392  —.299  Vanillic  —.017  .125  .557  .178  Syringic  .342  .354  —.498  —.194  p—Cournaric  .290  .138  —.704  —.114  Ferulic  .470  .275  —.772  —.316  89  The presence of salal was most highly correlated with the presence of p—hydroxybenzoic,  ferulic and syringic acids.  The  presence of all other herbaceous and shrubby vegetation was most highly correlated with p-hydroxybenzoic and syringic acids.  The  woody horizons were highly positively correlated with vanillic acid only, and highly negatively correlated with all other acids. Site type was not highly correlated with phenolic acids, but the negative values indicate that HA tended to have lower concentrations of phenolics than CH site. The results indicate that vegetation type has a large influence on the concentrations of bound phenolic acids present. Salal,  and possibly other shrubs,  such as Vaccinium (also  ericaceous), have waxy, thick cuticles that provide a source of coumaric and ferulic acids. syringic acids.  Wood,  The angiosperms also contribute  on the other hand, has very few phenolic  acids other than vanillic acid, a lignin derivative.  4.0  SUMMARY Significant differences were found in physical and chemical  properties between CH and HA sites. tend to occur side by side,  Although CH and HA sites  stand structures, humus profiles,  soil properties and moisture regimes are different.  HA sites  tend to occur on ridgetops, with CH sites adjacent on lower slopes.  The topographic position is correlated with moisture  gradients, being driest at the top and wetter towards the bottom. The CH stands therefore occupy a wetter site.  90  The higher topographic position also makes the site less protected from strong winds, making the HA stands more susceptible to windthrow.  And once windthrown, the stand  structure of the resulting stand would tend to further its’ susceptibility to windthrow.  The undisturbed CII stands are  relatively open and uneven—aged, which encourages windfirmness; in contrast, the HA stands are dense and even—aged, which encourages windthrow (Oliver and Larsen,  1991).  Windthrow  disturbances are very important in causing site differences. In terms of differences in understorey vegetation, the relatively open stand structure occurring in the CH phase, supports abundant and dense growth of salal.  Other vegetation  including Vaccinium spp., Menziesia ferruginea, Blechnum spicant and Cornus canadensis, are much less abundant.  The dense  structure of the HA stands supports only sparse shrub cover, including Vaccinium spp., and Blechnum spicant.  Mosses such as  Hylocomium splendens, Kindbergia oregana and Rhytidiadeiphus loreus occur on both phases. The CH phase, which is thought to be undisturbed for over 1000 years, was found to have almost continuous root restricting layers such as compacted or cemented horizons, a high occurrence of standing water over some portion of the year and gleyed mineral soil horizons. activity,  The HA had more visible windthrow  including a greater proportion of woody humus and  correspondingly deeper forest floors, more friable mineral soils, and deeper rooting zones than the HA. 91  This suggests that  windthrow disturbance is important in physically rejuvenating the site by breaking-up hardpans, mixing organic matter with mineral soils which increases the soil friability and encourages deeper rooting by enriching the mineral soil fertility. major disturbance events,  Thus, without  such as windthrow, CII sites would tend  to degrade further with a greater occurrence and build—up of Hhi humus horizons, cemented horizons,  and standing water.  Humus profiles of the CH and HA phases were found to have six predominant humus horizons.  Three of these horizons were of  woody origin, ranging from the least decomposed Fw horizon, to the residuic Hrw, to the well decomposed Hw.  The remaining three  horizons were of non—woody origin, ranging from the least decomposed Fm horizon,  to the well decomposed Hh, and an Hhi  which was an organic horizon containing intermixed mineral particles found immediately above the mineral soil.  The majority  of fine roots were found in the Fm, Hh and Hw horizons. The windthrow process was responsible for the deposition of a large amount of woody material on HA sites.  Poorly decomposed  woody horizons (Fw and Hrw) were found to occur on 68% of HA plots, but only 38% of CII plots.  Well decomposed Hw horizons  were found on 56% and 38% of HA and CH plots respectively. they did occur,  Where  there was not significant difference in depth of  woody horizons between CH and HA.  The non—woody horizons Fm, Hh  and IIhi were deeper and occurred more often on the CII plots. Humus profiles also reflected differences due to windthrow. The HA was found to have 38% Hemimors  92  (consisting of thin F and H  horizons occurring over a windthrown mixture of organic and mineral soil),  and 41% Lignoxnors (consisting of greater than 35%  decaying wood),  but only 22% Humimors  undisturbed organic horizons).  (reflecting relatively  In contrast, the CR had more  diverse humus profiles including 57% Huinimors, Hydromors  (which develop under fluctuating,  17% Lignomors,  stagnant water)  24%  and  2% Hemimors. Nutrient concentrations were found to vary between horizons. Not surprisingly, the woody humus was found to be significantly lower than non—woody humus for pH, and P,  total N and S, available N, S  and total inorganic K and Mn.  Nutrient concentrations  tended to increase with increasing decomposition of woody horizons.  The 11w horizon was found to have abundant fine roots,  and was thus the most biologically important of the woody horizons.  Nutrient concentrations within the 11w horizon were  found to differ between CII and HA sites;  the pH, moisture  content and C/N ratio of those from the CH were higher than those from the HA,  while total N and S,  and available N and P were  significantly higher than those of the HA compared to the CR. This indicates that the CR site seems to be decomposing either at a slower rate than the HA,  or that decomposition is less  complete. For non—woody horizons, the Fm horizon was found to be significantly higher than other non-woody horizons in available P and exchangeable K and Mn.  There were no significant differences  in nutrient concentrations between Fm and Rh horizons. 93  The Hhi  horizon, occurring close to the mineral soil interface, had significantly higher ash contents, Fm horizon,  and total C, N and S.  For the  samples from the CH sites were found to be  significantly higher than from the HA for pH, moisture content, available S, and exchangeable Ca, K, and Mn; this most likely reflects differences in litter input between the sites, and to For the Hhi horizon,  the wetter conditions of the CH.  samples  from the CH sites were found to be significantly higher than for the HA for pH and water content, but available P was higher for HA than for CE. Lipids concentrations were found to increase with increasing decomposition, while total and labile polysaccharides and cellulose were found to decrease with increasing decomposition. Lipids, total and labile polysaccharides and cellulose were all found to be higher for CH horizons than for HA.  This suggest, as  with the nutrient concentration differences between sites, that the CH site seems to be decomposing either at a slower rate than the HA,  or that decomposition is less complete.  A comparison of bound phenolic acid concentrations between humus horizons found that woody horizons tended to be higher in vanillic acid while the non-woody horizons tended to be higher in protocatechuic, p—coumaric and ferulic acids.  The Fm horizon,  with the greatest proportion of angiosperm inputs, had the highest concentrations of protocatechuic acid, degradation product of angiosperm lignin), ferulic acids  syringic acid (a  and p—coumaric and  (thought to originate from the cutin of leaf and  94  other plant tissues and from the suberin of roots); the Fw, which is pure coniferous wood, had the highest concentration of vanillic acid (a degradation product of coniferous lignin); the Hhi, was found to be highest in p-hydroxybenzoic acid. Interestingly, the Fm, which is the most biologically active horizon, was found to have the highest overall concentration of phenolic acids, while the Hhi, which would be expected to accumulate the mobile organic acids from overlying horizons, was found to have the second highest overall concentrations of phenolic acids. When the CH site, with characteristically abundant salal was compared with HA sites, with relatively sparse understorey vegetation, the CH site was found to be significantly higher in syringic, p—coumaric and ferulic acids.  The concentration of p—  hydroxybenzoic acid, originating from suberin of roots, was not significantly different between sites, but tended to be higher on CH sites than on HA.  5.0 CONCLUSION 5.1 Soil Classification Hypothesis: That distinct and recognizable humus horizons occur commonly on both the CH and HA sites, but that the relative abundance varies between the sites. Conclusion: Accept.  95  5.2 Chemical Differences in Humus Horizons Between CH and HA Hypothesis: That similar horizons from the CH and HA sites are different with respect to chemical composition including total and/or available nutrient concentrations,  lipid and  polysaccharide contents, and phenolic acid content. Conclusion:  Accept that some available nutrients,  lipids  and polysaccharides and phenolic acid contents are different between some horizons from CH and HA sites.  96  Chapter IV Chemical Characterization of Humus Horizons Using 13 Carbon Nuclear Magnetic Resonance’  1.0  INTRODUCTION  Nuclear magnetic resonance (NMR)  spectroscopy is a technique  originally developed for organic structure determination in chemistry.  However, since 1970, developments in NNR have  facilitated its application to many areas of applied science, including soil science.  Solid-state 13 C NMR has recently been  used to characterize forest litter and humus layers 1988; Kogel-Knabner humification  (Hempfling  J.,  1988), j.,  (Kogel  litter decomposition and  1987; Hammond g  1985).  Other applications of ‘ C NMR in soil science and forestry are 3 listed in Preston and Rusk (1990).  A review of the NNR technique  is beyond the scope of this thesis but is well described by Wilson (1987) This study used ‘ C NNR spectroscopy to examine chemical 3  1  This chapter is based on the publication by de Montigny (1992). 97  differences in the humus types between the ecosystem phases which could explain the differences in forest productivity after clearcutting.  2.0  METHODS 2.1. Sample Preparation Samples representative of the 6 humus types from the 2 site  phases  (as described previously) were collected,  ground to pass a 20 mesh sieve.  In addition,  air dried and  coniferous litter  and salal litter were collected from each of the 5 CII and/or HA sites,  air—dried, bulked and ground to pass a 20 mesh sieve.  Replication was not possible due to the high costs of 13 C NNR. However, the excellent agreement between samples of similar humus between sites indicates that choosing representative humus types from the CH and HA sites provided adequate sampling for this study.  2.2 NMR Spectroscopy The NNR spectroscopy was done by Dr. Patrick G. Hatcher of Pennsylvania State University.  Dry samples were packed into a  bullet-type rotor that was placed in the probe of a Chemagnetics Inc. M-100 NMR spectrometer operating at an 1 H field of 100 MHz. The sample, which was spun at a rate of approximately 3.5 kHz at the “magic—angle” of 54.7  °  to the magnetic field, was analyzed  by the technique of cross polarization with magic-angle spinning as described by Hatcher (1987).  Dipolar—dephased (DD) 98  CPMAS  spectra were generated by inserting a delay period of 40-100 js without 1 H decoupling between the cross—polarization and acquisition portions of the CPMAS pulse sequence Frey,  1979).  (Opella and  Chemical shifts are reported relative to  tetramethylsilane  (TMS)  at 0 ppm.  2.3 Spectral Analysis  Spectra were divided into chemical shift regions according to chemical types of C as follows: A, aliphatic 0-50 ppm; B, methoxyl 50-60 ppm; C, 0-alkyl 60-96 ppm; D, di-O-alkyl and aromatic 96—141 ppm; E, phenolic 141-159 ppm; F, carboxyl 159-185 ppm; and G,  aldehyde and ketone 185-210 ppm.  In the context of  this paper, the term aromatic C is used to designate, specifically, the nonoxygenated aromatic C occurring at 96—141 ppm,  and the term phenolic is used to designate the oxygen-  substituted carbons at 141-159 ppm. Areas of the chemical shift regions were measured by cutting and weighing the spectra and are expressed as percentages of total area  (relative intensity).  The proportions of lignin and  carbohydrate C were then determined using the procedure described by Preston  al.  (1990).  Briefly, these include:  Total Lignin Carbon CL  =  4.5 E + B  Total Carbohydrate Carbon =  1.2  (C  —  99  1.5E)  Ratio of Carbohydrate to Lignin Monomers Cm Lm  1.2 (C 3E  =  l.5E)  —  Aromatic Lignin Ar*  =  D  —  0.2  (C  —  1.5E)  Ratio of Carbohydrate to Aromatic Lignin Monomers =  Lm  1.2 (C 1.5E) E+Ar* —  The problems and uncertainties inherent in analyzing the spectra this way have been described by Preston  .  (1990).  The most notable is that there are problems with peak overlap and lack of completely specific chemical shift regions,  for which the  use of vertical divisions and correction factors cannot fully compensate.  Thus the analysis of lignin signals is based only on  the guaiacyl structural unit, which is a major component of coniferous lignin.  This is satisfactory for the woody horizons,  Fw and Hrw, which are almost exclusively the carbohydrate— depleted residue of coniferous wood with little alteration of lignin structures.  The results are less meaningful for horizons  in which decomposition has proceeded further, resulting in alteration of lignin structures and in non—woody horizons derived from salal and needle litter.  Therefore, these calculations were  not done for the Hhi horizons.  3.0  RESULTS AD DISCUSSION 3.1 Carbon-13 CPMAS NMR Characterization of Humus Horizons  100  3.1.1 Woody horizons To aid in the interpretation of the NNR spectra, the structural units of cellulose, guaiacyl lignin and condensed tannins are shown in Figure 4.1.  Spectra of woody and non—woody  horizons from CH and HA sites are shown in Figures 4.2 and 4.3, respectively, and the relative proportion of C in the chemicalshift regions in Table 4.1.  The general features of the spectra,  and the values in Table 4.1,  indicate that there is little to  distinguish samples taken from the CH and HA sites.  There are  also only small differences between Fw and Hrw spectra, but a larger difference between this pair and the Hw spectra. The spectra of the Fw and Hrw horizons are similar to those reported previously for well—decomposed gymnosperm wood (Preston 1990;  Hatcher,  1987).  These spectra  (Figures 4.2 a—d)  are dominated by signals typical of the guaiacyl lignin unit (Figure 4.lb) which predominates in softwoods; these include methoxyl C at 56 ppm, ppm.  and aromatic and phenolic C at 110 to 160  The phenolic region (141-159 ppm)  at .148 ppm,  free C -OH at 146 and C 4 4 participating at C-O-C 4 ether  linkages at 153 ppm CLeary (96—141 ppm)  includes the guaiacyl C 3  j.,  1986).  arises from guaiacyl C , C 1 , 2  (including carbohydrate)  The aromatic region 5 and C C . 6  0—alkyl C  is depleted in these spectra, due  largely to the three—C side chain of lignin. In fresh wood, the 0-alkyl region (and in fact, the whole spectrum)  is dominated by signals from carbohydrates, mainly  cellulose (Hemmingson and Newman, 101  1985; Newman and Hemmingson,  (A) cellulose repeaLing unit  (B) lignin repeating unit  OH 2 ecjH  H H—  _o_?\gH/  H  H—  OH  H— COH  1 R  2 R (OH 0  —.  1 R  C  =  OCH3, R 2= H  gualacyl  =  3 2 = OCH R  syringyt  =  2= H R  phenyipropane  (C) condensed tannin repealing unit  OH  R R  Figure 4.1.  = =  H OH  procyanidin unit prodeiphinidin unit  Structural units of a) condensed tannins. 102  cellulose, b)  lignin and c)  5 5 4 4 5 5  20 21 19 18 38 25  Hw  Hrw  Fw  CH HA  CH HA  CH HA  23 17  14 10  8 11  7 5  9 10  10 11  5 4  Methoxyl (50—60)  26 27  Woody Horizons  Litter CH HA Fm CH HA Hh CH HA Hh i CH HA  Non—Woody Horizons  Aliphatic (0—50)  24 24  24 25  27 23  20 20  28 27  32 29  33 44  o-Alkyl (60—96)  103  27 28  33 34  34 35  18 24  27 27  24 24  19 14  11 13  14 14  15 15  8 11  11 10  10 10  9 6  Aromatic Phenolic (96—141) (141—159)  6 8  4 4  3 3  8 10  8 9  6 7  6 4  Carboxyl (159—185)  3 5  3 3  3 2  4 5  5 4  3 4  1 1  Carbonyl (185—210)  Table 4.1.: Relative percentages of carbon in chemical shift regions (ppm) of humus types by site.  Table 4.2: Ratios of calculated total lignin (Liii), carbohydrate (Cm) and aromatic (Ar) carbon and associated ratios Cm/Lm* is calculated using for humus types by site. Ar.  Lm  Cm  Ar  Cm/Lm  CmILrn*  Non—Woody Horizons Litter CH HA Fm CH HA Hh CH HA Hhi CH HA  43.6 30.9  24.5 41.5  15.1 7.5  1.1 2.3  1.0 3.1  48.1 48.7  21.1 17.2  25.7 18.2  0.7 0.6  0.6 0.6  52.8 50.0  13.3 14.4  21.0 21.5  0.4 0.5  0.4 0.5  39.4 54.8  10.1 4.4  14.0 20.0  0.5 0.1  0.4 0.1  CH HA  78.7 80.2  4.7 0  28.6 30.1  0.1 0  0.1 0  CH HA  70.0 74.5  4.6 4.2  28.3 28.8  0.1 0.1  0.1 0.1  CH HA  54.5 62.6  9.2 5.7  22.3 23.1  0.3 0.2  0.3 0.2  Woody Horizons Fw  Hrw  Hw  104  Figure 4.2.  120  60  0  -60  PPM 240 180  I  120  I  60  I  105  Carbon-13 CPMAS NNR spectra of woody horizons HA sites.  PPM 240 180  I  (d)Hrw  (c) [trw  I  (b)Fw  (a)Fw  I  HA Site  CII Site  0  I  (Fw, Hrw, Hw)  -60  I  from CH and  1990; Preston ppm)  1990).  and non-crystalline  anomeric C.  These include the crystalline (65  (84 ppm)  components of C , and the 4  Total carbohydrate C was calculated to be about 4%  for the less-decomposed Fw and Hrw, and 7.5% for the Hw (Table 4.2).  The ratio of carbohydrate to lignin moieties  in the range of 0.1 to 0.3.  (Cm/Lm) was  These are similar to values found  for highly—decomposed logs of Douglas—fir, western red cedar and western hemlock.  By contrast, ratios of 2—3 are found for fresh  wood (Hemmingson and Newman,  j.,  1985; Preston  1990).  Also in contrast to fresh wood, there is a broad region of intensity in the aliphatic region (0—50 ppm). from about 10% in the Fw Hw (Table 4.1).  ,  to 12% in the Hrw,  This increases and to 20% in the  There is also an increase in resolution; for the  Fw spectra, the aliphatic region is a broad shoulder most likely due to the selective preservation of waxes and resins in the original wood.  The Hrw spectra begin to show a peak at 30 ppm,  which is better defined for the CH site and for the Hw spectra; this region has a strong, well-defined peak at 30 ppm.  The peak  at 30 ppm is characteristic of aliphatic -CH -units in long 2 chains,  such as fatty acids, and is typically seen to increase  with increasing decomposition in Folisols and Histosols 1985; Preston  1987;  1989).  (Hammond  It is thought to be  largely a byproduct of production of microbial biomass coupled with incomplete decomposition.  The increase in 0-alkyl,  aliphatic and carboxyl C may be the result of microbial or fungal activity (Baldock  1990; Preston and Ripmeester, 106  1983).  There is a greater contrast between the Fw and Hrw spectra, vs. the Hw.  The Hw have more intensity characteristic of  carbohydrates, at 62,  73 and 101 ppm.  The carboxyl and carbonyl  intensity also increase from Fw to Hrw to Hw; this could be due both to oxidation of lignin and to the production of fatty acids in microbial biomass.  Decomposition of lignin,  other aromatic structural units,  or production of  is also consistent with a  relative decrease in methoxyl,  aromatic and phenolic C, and a  change in the aromatic region,  as the intensity at 130 ppm  increases relative to that from 115-125 ppm. calculated to be about 80% for the Fw,  Total lignin C is  72% for the Hrw,  and 58%  for the Hw (Table 4.2). It is not easy to ascertain the origin of the 0—alkyl intensity in the Hw spectra;  it may arise largely from the  original sugars in woody and non-woody litter input, be greater contribution from microbial activity.  or there may  However, the  usual pattern in organic soils is for plant—derived carbohydrates to decrease, while microbial aliphatic C increases  al.,  1985; Hempfling  Preston et al.,  1987;  j., 1989;  (Hammond  1987; Kogel—Knabner Zech  good resolution in the spectra,  j.,  1990).  1988; This, and the  suggests that the 0-alkyl C most  likely derived from the original inputs.  3.1.2  Non—woody horizons  The spectra of the non-woody Fm and Hh horizons  (Figure 4.3)  are similar to those reported for forest litter layers under 107  Figure 4.3.  60  I  0  -60  I  PPM 240 180 120  60  108  Carbon-13 CPMAS NMR spectra of non-woody horizons HA sites.  PPM 240 180 120  I  (I) Hhi  (e)HhI  I  (d)Hh  (c) Hh  I  (b)Fm  (a)Fm  I  HA Site  CII Site  -60 (Fm, Hh,  0  Hhi)  from CH and  conifers  (Kogel  1992;  1988; Krosshaven  Zech  .,  1987).  0-alkyl carbon (Table 4.1),  1990; Preston  The dominant signal is that from  indicating that polysaccharides are  quantitatively the most significant compounds.  0-alkyl C  accounts for 33 to 44% of the total C in the litter, Fm horizon,  30% in the  27% in the Hh horizon and 20% in the Hhi horizon  (Table 4.2).  The aromatics are the next most significant  compounds accounting for 14 to 19% of the total C in litter, 24% in the Fm horizon,  27% in the Hh horizon and 18 to 24% of the  total C in the Hhi horizons. cutin,  Aliphatics, which would include the  suberin, and highly aliphatic polymers of plant cuticles,  account for about 26% of the total C in the litter, the Fm and Hh,  and 25 to 35% in the Hhi.  18 to 22% in  Compared to the woody  samples, the methoxy C and phenolic C content is lower,  4 to 5%  and 6 to 11%, respectively. From the estimates in Table 4.2 for these non—woody horizons, total lignin increases with decomposition from Fm to Hh, while total carbohydrate C decreases.  The resulting ratio of  Cm/Lm decreases from a high of 2.3 in HA litter to approximately 0.5 for the Hh (values were not calculated for the Hhi horizon). The overall effects of decomposition of the non—woody horizons are a decrease in easily—decomposable 0—alkyl C, which would mainly be due to carbohydrates of plant origin, and an increase in aliphatics and carboxyls, most likely derived from residual plant material or microbial biomass. There is also some evidence for tannins in the spectra of 109  the non—woody horizons. most litter materials  Tannins are present in low quantities in  (Kogel-Knabner  p1., 1991), but are  difficult to identify in NMR spectra because the peaks occur in the same regions of those of lignin and carbohydrate (Czochanska 1980; Morgan and Newman,  1986; Preston and Sayer,  1992).  However, the peak due to C ’ and C 3 ’ of condensed tannins 5 (procyanidins and prodeiphinidins, ppm,  Figure 4.lc)  occurs at 144  in a region that is usually clear in wood and litter  spectra.  As discussed above, the phenolic region of the Fw  horizon from both CH and HA (Figure 4.2)  shows a sharp,  intense  peak at 149 ppm, with a light shoulder at 153 ppm, typical of guaiacyl lignin C 3 and C . 4 site  The Fm and Hh horizons from the CH  (Figure 4.3), which would have both coniferous and salal  litter inputs, show a broad signal combining the tannin peak at 145, the guaiacyl peak at 148, and the syringyl peak at 153. The peak at 144 ppm is less prominent in the HA than in the CH sites. It also decreases from Hh to Hhi for both sites, possibly from decomposition or complexation of tannins as they are leached through the soil profile. 3.2 Dipolar-dephased Carbon-13 CPMAS NMR Characterization of HUmUS Horizons  This technique can be used to examine features that may be masked in the normal CPMAS spectra. a signal acquisition,  During a delay period before  intensity is lost more quickly from carbons  that have strong dipolar interactions with protons; with directly bonded hydrogens.  i.e., carbons  The dipolar interaction is 110  weakened in two cases:  for nonprotonated C which have a greater  separation from hydrogen nuclei, motion in the solid state.  and where there is molecular  This occurs for methyl C due mainly  to methyl group rotation, while long-chain aliphatics can also display sufficient backbone vibrations to weaken the dipolar coupling in the solid state (Opella and Frey,  1979; Wilson,  1987) Dipolar-dephased spectra have proven useful in detecting tannins,  as the non—protonated C 4 and C 8 at 108 ppm can be  observed in DD spectra in a region that is otherwise masked by aromatic and anomeric CH (Wilson and Hatcher,  1988). This  provides a useful test for tannins in complex matrices,  for which  the only interference is the ketal C of carbohydrate, which would not likely be a problem for litter and soils. For the woody horizons,  the DD spectra for the Fw and Hrw  horizons in Figure 4.4 a-d show typical lignin peaks for methoxyl at 56 ppm, phenolic at 148 ppm with a shoulder at 153 ppm, nonprotonated aromatic C  ) 1 (guaiacyl C  weaker signals due to carboxyl (Hatcher,  1987; Preston  weak or absent.  J.,  at 132 ppm, as well as  (172 ppm) 1990).  For the Hw horizons  and  and carbonyl  (195 ppm)  Tannin signals are very  (Figure 4.4e and f), there  is some intensity at 108 ppm, but without the other characteristic tannin peak at 144 ppm.  There is also  considerable residual aliphatic intensity for both CH , 3 2 and Cl! consistent with the presence of long—chain hydrocarbon moieties with considerable molecular motion in the solid state. 111  C  Figure 4.4.  I  180 120  I  60  I  0  I  -60  I  PPM 240  I  I  180 120  I  60  0  -60  112  Dipolar-dephased Carbon-13 CPMAS NMR spectra of woody horizons from CH and HA sites.  PPM 240  I  (d) Hrw  (c) Hrw  (1)11w  (b)Fw  (a)Fw  -  HA Site DU  CHSite-DO  (Fw, Hrw, Hw)  Figure 4.5.  180 120  60  0  -60  PPM 240  I  180 120  I  I  0  I  60  -60  113  Dipolar-dephased Carbon- 13 CPMAS NMR spectra of non-woody horizons(Fm, Hh, Hhi) from CH and HA sites.  PPM 240  I  (f) Hhi  (e) HM  I  (d)Hh  (c)Hh  I  (h) Fm  (a)Fm  I  HA Site-DO  -  CH Site DO  For the non—woody horizons, the DD spectra of the Fm and Hh horizons from the HA site (Figures 4.5 a-d)  lack a clearly-  defined peak at 145 ppm, although there is some intensity at 108 ppm.  The tannin signal appears to be stronger for the CH  samples; however, the differences are small.  Quantitative  analysis would require a series of DD spectra with different dipolar dephasing times  (eg, Hatcher,  1987; Wilson,  well as analysis of replicate samples.  1987), as  Dipolar—dephased spectra  of the most decomposed Hhi samples indicate much lower tannin contents, spectra  consistent with the trend shown in the non—dephased  (Figure 4.3e and f).  As was seen for the DD Hw spectra,  those from the non—woody horizons also show considerable residual intensity from more mobile long-chain aliphatic C.  3.3 Carbon-13 CPMAS NMR Characterization of Litter Inputs To trace the source of the tannins in the organic horizons, some litter inputs and other salal plant parts were examined. The CH and HA litter (Figures 4.6b and c)  are similar to  coniferous litter reported elsewhere (Norden and Berg, Preston  1992;  Zech  1987),  1990;  as well as to the Fm  horizons except that the resolution is better as there has not been any decomposition.  Tannins,  if present are only indicated  by the breadth of the phenolic signal with poorly resolved intensity at 145 ppm. different,  The salal litter  (Figure 4.6 a)  is  as it clearly shows a tannin peak at 144 ppm.  spectra of salal flowers,  leaves and roots, shown in 114  The  (a) Salal Litter  (b) CH Litter  (c) [IA Litter  I  PPM 200 Figure 4.6.  I  I  ——  100  I  I  0  Carbon-13 CPMAS NMR spectra of litter materials from: a) CH site salal, b) CH site coniferous litter, and c) HA site coniferous litter. 115  (a) Salal Flowers  (b) Salal Leaves  (c) Salal Roots  F---  PPM 200  Figure 4.7.  100  0  Carbon-13 CPMAS NNR spectra of salal: b) leaves and c) roots. 116  a)  flowers,  Figure 4.7,  all have a strong tannin signal at 144 ppm.  Interestingly, the tannin content in the leaves is less than in the flowers and roots.  Furthermore, the same peak is clearly  seen in the litter (Figure 4.6a),  indicating as expected, that  the tannins do not readily decompose. A sample of salal leaf tannin was run using solution NMR , 2 (Preston  1991, pers. comm.).  The spectra was similar to that  of proanthocyanidins reported by Czochanska  (1980).  More  specifically, the spectra appears to be that of a polymer of procyanidin and prodelphinidin units  (Figure lc).  3.4 Site Differences The organic components of similar horizons from the two site types did not show any unusual features that would explain dramatic differences in seedling performance between the sites. That is, the woody horizons were found to be similar to large gymnosperm logs decomposing on the forest floor as seen in other Similarly, the non—woody horizons are similar to those  studies.  widely reported for Histosols and forest organic horizons. However, one major difference is that tannin signals appear to be stronger in CH samples than in HA.  This may be due to salal,  which was found to have strong tannin signals relative to coniferous litter. The presence of tannins on the CH site may explain some of  2  Dr.  C.  Preston, Pacific Forestry Centre, Victoria, B.C. Canada 117  Forestry Canada,  the differences in forest productivity following clearcutting. Tannins have been found to reduce the biodegradability and humification of organic matter by three processes; the production of protein—tannin complexes which are much more resistant to microbial decomposition than unaltered proteins; the permeation and coating of nonproteins such as cellulose and hemicellulose by the protein—tannin complexes, giving them considerable resistance to microbial attack; and by the inactivation of enzymes important in the process of decomposition (Benoit and Starkey,  1968a,b).  Other factors which may be affecting decomposition is the wetter site conditions of the CH (as previously discussed),  and the  higher concentration of aliphatics, particularly of lipids  (as  previously discussed). Small differences seen in the NMR spectra between the CH and HA may reflect differences in decomposition between the CII and HA phases.  Litter samples from the HA site have a much higher  carbohydrate content than that from the CH sites.  Yet,  in the Fm  horizon, the HA has less carbohydrate than the CH site.  In fact,  total carbohydrate C,  in both the woody and non—woody horizons  tend to be higher on the CII, while total lignin C tends to be lower (Table 4.2).  The ratio of total carbohydrate to lignin C,  also tends to be higher on the CII.  All of this seems to indicate  that the carbohydrates on the CII site tend to be more resistant to decomposition than on the HA sites.  Furthermore, the lower  availability of nitrogen and phosphorus on the CH site, relative to the HA site, as found in Chapter 3, may be due to differences 118  in decomposition.  Tannins from salal litter,  or aliphatics  peculiar to salal or western red cedar litter, or the wetter site conditions may be factors important in causing this resistance to decomposition.  4.0 SUMMARY  Humus horizons identified in the study had an excellent correspondence with spectra of ‘ C NNR. 3 dominated by signals from lignin,  Woody horizons were  but with increasing  decomposition, the relative proportion of lignin decreased, while aliphatics and carbohydrates increased, presumably from microbial and fungal sources.  The non—woody humus types were typical of  forest litter layers, which are dominated by signals in the carbohydrate region.  Increasing decomposition resulted in  decreasing carbohydrates and increasing aliphatics and carboxyls. There were no apparent differences between the CH and HA sites, with the exception of the presence of tannins.  Spectra from  salal leaves and litter were found to contain tannin compounds. These tannins were also found in the Fm horizons on both CH and HA sites, but intensity was greater on samples from the salal— dominated CH sites.  The ratio of total carbohydrate to lignin C  tended to be higher for the CH humus horizons,  indicating that  the carbohydrates in these horizons may be more resistant to decomposition, possibly as a result of the tannins, or of aliphatic compounds peculiar to vegetation on the CH site, or of the wetter soil conditions on the CH. 119  These factors could be  important to the overall reduction of forest productivity seen on salal-dominated CH sites.  5.0  CONCLUSION  1)  Hypothesis: That the 13 C CPMAS NMR spectra obtained from the different humus horizons are distinct and recognizable. Conclusion: Accept that the different humus horizons identified are distinct and recognizable.  2)  Hypothesis: That similar horizons from the CH and HA sites are different with respect to phenolics and tannins. Conclusion:  There is evidence to suggest that the litter  and F horizons on CH sites may have a higher tannin content, but more work is needed to determine actual concentrations before the hypothesis can be accepted.  120  Chapter V  Allelopathic Potential of Salal  1.0  INTRODUCTION Ericaceous plants have long been known to contain relatively  high concentrations of phenolic acids  (Harborne and Williams,  1973), many of which are known to be allelopathic  (Rice,  1984).  Many studies suggest that phenolic acids produced by ericaceous species could adversely affect the growth of competing vegetation including grasses,  shrubs and trees.  As discussed in Chapter 2,  these studies include that of Arctostaphyllos glandulosa in California Spain  (Muller et al.,  (Ballester  and Cuervo, (Handley,  1980)  1968),  Erica species in Northwest  .]., 1977; Carballeira, 1980; and Carballeira Calluna vulgaris in the United Kingdom  1963; Jalal  al.,  1982; Jalal and Read,  Kalmia angustifolia in Eastern Canada  (Mallik,  1987)  hermaphroditum in Sweden (Zackrisson and Nilsson, Salal may also have an allelopathic effect.  121  1983a and b), and Empetrum  1989). Towers  (1966)  found salal to contain the phenolic acids p—  hydroxybenzoic, o—pyrocatechuic, gentisic, protocatechuic, vanillic,  syringic, p—coumaric, caffeic,  (Towers  acids  pJ.,  1966).  ferulic and sinapic  The tannins  (polyphenolics)  found  to be present in the leaves, roots and litter of salal using ‘ C 3 NMR,  as described in the previous section,  potential allelochemicals.  could also be  The fatty acid concentration in  ericaceous plants is extremely high,  and some breakdown products  such as nonanoic acids are known fungitoxins Robinson,  1969),  phytotoxins (1971)  (Garrett and  and octanoic and decanoic acids are known  (Overbeek and Blondeau,  1954).  Del Moral and Cates  found some evidence for allelopathy by salal in field  bioassays using litter extracts on germination of barley (Hordeum vulgaris)  and an annual grass  (Bromus tectorum).  Phenolic acids as allelochemicals were the focus of this study, primarily because of ease of access to a High Performance Liquid Chromatograph used exclusively for phenolic acids.  The  presence of free phenolic acids in soil solution was felt to better represent natural conditions,  since alkali extraction, as  is commonly used, may release phenolic acids by degradation of organic matter 1978).  (Whitehead  J., 1981; Kaminsky and Muller,  Whether the phenolic acids found in soil solution, or the  polyphenolics in salal leachates occur in sufficient concentrations in the soil solution to affect conifer growth remains a critical question. This study was initiated to determine: 122  1)  the seasonal  concentration of the free phenolic acids in soil solution under vigorously growing salal,  including p-hydroxybenzoic,  protocatechuic, vanillic,  syringic, p—coumaric and ferulic acids;  the effects of the phenolic acids at field concentrations  and 2)  and of leachates from the flowers and fruits of salal plants on the germination, growth and uptake of radioactively-labelled phosphorus in three species of conifers:  Sitka spruce, western  hemlock and western red cedar.  2.0  METHODS 2.1 Seasonal Phenolic Acid Concentration 2.1.1 Soil sampling The plantations selected for sampling of free phenolic acids  were located in Western Forests Products T.F.L.  (aged 4 years since logging,  McNeill at Misty 100 and 400 slashburning and planting),  and Rupert 200 and 418  slashburning and planting).  since logging,  42 near Port  (aged 8 years  Soil samples were  taken from beneath vigorous young salal plants in February, May, July,  September and November,  period,  1990.  In the February sample  four samples were collected from each plantation, two of  woody origin (including Fw, Hrw and Hw) origin (Fm).  and two of non-woody  Because of high variability in phenolic  concentrations,  it was decided to double the sample number,  so in  subsequent sampling periods eight samples were collected from each plantation, origin.  four of woody origin,  and four of non—woody  Samples were left overnight in cool shade,  123  and  transported to the laboratory at the Pacific Forestry Centre the next day.  They were kept in cold storage at 4°C until processed  (within 1 week).  Immediately before centrifuging,  soils were  sieved through a 10 mm sieve to remove large roots and woody pieces, then through a 4 mm sieve to homogenize the sample.  A  small sample, approximately 20 g, was taken to determine water content at 70°C.  2.1.2 Sample preparation  Fresh,  sieved soils were centrifuged at 10,000 r.p.m.  minutes to extract the soil solution.  for 20  This method used 30 cc  syringes with the handle sawed of f and an 8 cm piece sawed directly beneath to be used as a “syringe support”,  so that the  syringe support and shaft fit perfectly into the centrifuge tube (see Figure 5.1).  A small circle of filter paper was cut to fit  at the base of the syringe shaft, small amount of polyester wool  and this was covered with a  (used for fish tank filters).  tube was then weighed and filled with soil.  The  Approximately 40 g  of wet soil could be centrifuged per tube, with 8 tubes centrifuged per run,  and 2 runs per sample,  for a total of about  300 g of wet soil centrifuged for each sample.  The soil solution  was poured out of each of the centrifuge tubes into plastic screw—cap cups,  and the bulked sample was then kept frozen at  —  30°C until shipped to the University of British Columbia Soil Chemistry Laboratory for phenolic acid analysis. On thawing, the solution was measured for pH, 124  acidified to  125  t  Filter paper  Syringe  Centrifuge tube  Figure 5.1: Design of soil centrifuge tubes modified from a 30 cc syringe.  30cc Syringe  Support  Handle  pH 1.0 with 6 N HC1,  centrifuged at 6000 rpm for 15 minutes, then  filtered on Whatman #1 filter paper.  In a 500 ml separatory  funnel the solution was extracted three times with 75 mis of glass distilled di-ethyl ether.  Anhydrous 4 SO was added to 2 Na  the collected fractions in a 250 ml glass—stoppered flask, and left for 30 minutes.  This was then filtered (Whatman #1)  250 ml beaker and dried overnight.  into a  The residue was taken up in 2  ml of methanol for analysis by high performance liquid chromatography  2.1.3  (HPLC).  HPLC analysis  Prior to injection into the high performance liquid chromatograph, samples were filtered through a 0.2 micrometer Prep—Disc Filter (Biorad).  Chromatographic conditions used an  acetonitrile gradient as follows: Time 0 11 16 30 33 35 40 43  %  (l% Acetic Acid 92 92 86 86 40 40 92 92  % Acetonitrile 8 8 14 14 60 60 8 8  Flow = 1.5 mi/mm Wavelength = 280 nm Temperature = Room temperature Attenuation = 4 (free phenolics) Chart speed = 0.5 cm/mm Detector Sensitivity = 0.01 Standard = 2 ppm (free phenolics) Peak Threshold = 22 Peak Width = 6 Column = ODS Spheri-5 25 cm (Brownlee) 126  Guard Pak = Solvents = Standards  =  Bondapak C18 Inserts (Waters) Acetic acid Aristar (BDH) Acetonitrile Omnisolv (BDH) Protocatechuic, p—Hydroxybenzoic, Vanillic, Syringic, Trans—p—coumaric, Ferulic (Sigma)  2.1.4 Statistical analysis  Data obtained by HPLC was transformed from ppm (equal to mg per litre) soil)  to nanograms per gram of oven dry soil  (ng/g o.d.  as follows: mg/litre X .002 litre X hg o.d.soil X 1000000 ng/mg  where .002 litre is the volume of injected sample, g o.d.  soil is  the oven dry weight of soil centrifuged per sample, and 1000000 is the conversion factor from milligrams to nanograms. Results were analyzed using the statistics computing packages of SYSTAT (Wilkinson,  1990).  normality and homogeneity of variances.  All data were checked for Log transformations were  done if variances proved unequal using Bartlett’s test, then retested to ensure homogeneity.  Data for individual species were  analyzed separately. The analysis involved a one—way analysis of variance for individual phenolic acid concentration by date of sampling with Tukey HSD post hoc tests of pairwise mean differences and MGLH two way analysis of variance of phenolic acid concentration by date and humus horizon.  A correlation analysis was also done to  determine correlation between phenolic acid concentration and soil pH and moisture content.  127  2.2 Effects of Phenolics on Conifers  2.2.1 Seed germination Germination procedure was done according to International Seed Testing Procedures  1985).  (Anon.,  Seeds of Sitka spruce,  western hemlock and western red cedar were obtained from Pacific Forestry Centre, Victoria, at -18°C. River,  where they had been stored frozen  B.C.  The Sitka spruce seed was collected in 1988 at Salmon  the western red cedar in 1982 at Qualicum,  and the western  hemlock in 1979 at Salmon River. Seeds were given either no pretreatment,  or a stratification  treatment for 21 days at 0°C using distilled water.  Four  germination treatments were tested using the following solutions: 1)  a control using distilled water  2)  a phenolic acid solution consisting of protocatechuic, hydroxybenzoic, vanillic, acids  (BDH)  (pH 6);  syringic, p—coumaric and ferulic  at concentrations equivalent to maximum  concentrations found in natural soils under salal as follows  3)  (pH 5):  Phenolic Acid  nanomoles/1  protocatechuic hydroxybenzoic vanillic syringic coumaric ferulic  0.135 0.510 1.15 1.58 0.31 0.10  nanograms/l 21 70 193 313 51 19  an extract of salal flower and berries  (pH 4)  using 50 g  air-dried material per litre of distilled water, 24 hours and filtered  (#4 Whatman),  128  and  shaken  4)  a soil solution (pH 4)  obtained by centrifuging Flu horizon  (taken from under vigorously growing salal)  at 11,000 rpm  for 20 minutes.  Each treatment consisted of 4 replicates of 100 seeds each, germinated in 4 X 4 plastic germination boxes.  The seeds were  germinated on filter paper overlying layers of tissue paper moistened with 40 mls of the appropriate solution.  Sitka spruce  and western red cedar were grown for 21 days at 30°C  days and  20°C nights.  Western hemlock was germinated at a constant 20°C  for 35 days.  Germination was defined as the radicle reaching  four times the length of the seed coat. Data was analyzed using three indices of germination for normal germinants: 1)  50 value, the number of days to reach 50% of total R germination;  2)  Germination capacity (GC), the germination percentage after 21 days for Sitka spruce and western red cedar, and 35 days for western hemlock;  3)  Germination value (GV), X PV.  calculated by the formula GV  MDG (mean daily germination)  =  MDG  is the quotient  obtained by dividing the accumulated total number of germinants by the number of days of the test, (peak value of germination)  and the PV  is the maximum quotient  obtained by dividing daily the accumulated number of germinants by the corresponding number of days. 129  2.2.2 seedling growing conditions  Thirty—two germinants each of Sitka spruce, western hemlock and western red cedar germinated as described above were transplanted to flats of small pots.  Each pot contained 2  germinants, with rows of each species interspersed randomly throughout the flat.  Each flat consisted of one treatment.  The  soil media was a standard forestry mix consisting of 3:1 peat:verrniculite, with 2 kg lime/rn 3 of growing medium added. Germinants were grown in a growth chamber for 12 weeks with a regime of 16 hours of full incandescent and fluorescent light, temperatures of 21°C days and 18°C nights, and relative humidity of 60%. Seedlings were watered twice a week, once with distilled water,  and then with the 4 solutions described for the  germination experiment.  Seedlings were misted daily until seed coats  water each pot. were shed.  About 20 mls of solution were used to  When 80% of seed coats were shed (8 weeks),  seedlings  were fertilized with Green Valley Soluble 20-20-20 Plant Food (20% nitrogen, trace Fe,  20% phosphoric acid,  Cu, Mn,  Zn, B,  and Mb).  used in 8 litres of water,  and 20% soluble potash, and Two grams of fertilizer were  an equivalent of 100 ppm N.  The seedlings were harvested by carefully removing them from the growing medium. The roots were excised at the root collar, gently washed under running water and subsequently used for the seedling root bioassay as described in the next section. Following the root bioassay treatments, both roots and stems were 130  oven—dried at 70°C overnight and weighed to the nearest milligram.  2.2.3 Seedling root bioassay  The seedling root bioassay was carried out as described by McDonald  J., (1991).  Seedlings were harvested, the tops  clipped of f at the root collars, and the roots washed carefully under running tap water and kept under moist paper towels.  The  excised roots from 20 seedlings for each species were placed into cheesecloth bags.  One bag of each species was then immersed into  a solution containing 5 X l0 M calcium sulphate,  5 X l06 M  potassium dihydrogen phosphate and about 1 MBq 32 P per litre as orthophosphate.  Roots were left for 15 minutes, then immediately  washed for five minutes in running tap water to remove unabsorbed p from the root surfaces. 32  The washed roots were removed from  the cheesecloth, placed on aluminum trays, and dried at 70°C overnight. When dry, the roots were weighed then digested in a Technicon Block Digester using 5 mls of concentrated sulphuric acid and 1 ml of 30% hydrogen peroxide, completely digested.  for 3 to 6 hours until  Samples were allowed to cool before adding  10 mls of distilled water and pouring into scintillation vials. The 32 P content was counted as Cerenkov radiation in a Packard liquid scintillation spectrometer. The data converted from counts per minute (CPM) per minute (DPM)  to decays  by correcting for specific activity of the 131  uptake solution and background, quench and decay factors as follows: DPM  (CPM  =  -  B)  X 0.53 X decay  where DPM is the disintegration per minute,  CPM is the counts per  minute measured, B is the average background count of blanks, 0.53 is the efficiency of the scintillation counter,and decay is the radioactive decay of each count based on the reference date of the solution radioactivity. The specific activity of the uptake solution was then calculated by multiplying the DPM by the total P concentration (5 P X l0 N 4 P0 and dividing by the concentration of radioactive 32 2 K (220 X 106 DPM).  The uptake was expressed as moles of Pi.  2.2.4 Mature root bioassay  Root samples were collected from mature western red cedar and western hemlock in late February, Regional Park in Vancouver, B.C. British Columbia).  1991 in the Pacific Spirit  (adjacent to the University of  The site selected was a 75 to 80 year old  mixed western red cedar and western hemlock stand with a site index of 32 m at 50 years and a standing net merchantable volume of 750 m 3 per ha.  Root segments, usually mycorrhizal, of 10—20  cm axial length and 0.5—2.0 mm diameter were carefully removed from the 0—5 cm forest floor  (LFH) horizon.  These samples were  transported to the laboratory between moist paper towels in plastic bags. water,  The roots were washed carefully under running tap  and twenty roots of each species placed into each of 3 132  cheesecloth bags, which were then kept under moist paper towels until assayed (almost immediately).  One bag of each species was  then immersed into each of the 3 treatment solutions each containing 5 X  M calcium sulphate,  5 X 106 M potassium  dihydrogen phosphate and about 1 MBq 32 P per litre as orthophosphate.  Roots were left for 15 minutes, then immediately  washed for five minutes in running tap water to remove unabsorbed p from the root surfaces. 32  The washed roots were removed from  the cheesecloth, placed on aluminum trays, overnight.  These were digested,  and dried at 70°C  counted and analyzed as  previously described.  2.2.5  Statistical analyses  Results were analyzed by analyses of variance using the statistics computing packages of SYSTAT (Wilkinson,  1990).  All  data were checked for normality and homogeneity of variances. Log transformations were done if variances proved unequal using Bartlett’s test, then retested to ensure homogeneity.  Data for  individual species were analyzed separately. Germination data was analyzed by analysis of variance and Duncan’s multiple range test.  Seedling growth and root bioassays  were analyzed by one—way analysis of variance by treatment with Tukey HSD post hoc tests of pairwise mean differences.  133  3.0  RESULTS AND DISCUSSION Seasonal Phenolic Acid Concentration  3.1  The mean concentration of free phenolic acids in soil solution over a one year period for both woody and non—woody humus in nanograms per gram of oven—dry soil are presented in Table 5.1. et al.  The values are within the range reported by Whitehead  (1983).  Vanillic acid, hydroxybenzoic acid and syringic  acid were the most concentrated, accounting for 39%, of total measured phenolic acids respectively, protocatechuic, p—couxnaric and ferulic acids  26% and 19%  followed by  (accounting for 8%,  6% and 2% respectively). When yearly means of phenolic acids were compared between woody  (Hw)  and non—woody (Fm)  horizons, the non—woody horizons  were always significantly higher in phenolic acid concentrations than woody horizons, with the exception of hydroxybenzoic acid, which was not significantly different (Table 5.1).  This is  consistent with the results of the bound phenolic acids in Chapter 3. When the phenolic acids were compared over the 5 sampling periods,  significant differences occurred both between dates  sampled and horizons  (Table 5.1 and Figures 5.2 and 5.3).  Woody  horizons had no significant differences in phenolic acid concentration over the season for hydroxybenzoic, vanillic, p— coumaric or ferulic but differences were significant for syringic acid, which was highest in September and lowest in May, and protocatechuic acid, which was highest in May and lowest in 134  Vanillic  p-Hydroxy benzoic  Proto catechuic  water content  pH  Table 5.1:  Hw  Fm  Hw  Fm  Hw  Fm  Hw  Fm  Hw  Fm  135  13.4 abc* 6.5 a (8.4) (8.6) 6.0 a 7.0 a (6.7) (6.3)  18.6 bc* (9.8) 9.5 a (8.8)  41.0 c* (66.6) 9.0 a (4.5)  5.2 a (3.0) 4.0 a (3.0)  1.4 a (2.4) 0.6 a (1.1)  3.3 abc* (2.2) 1.4 ab (1.8) 5.0 a (4.2) 6.3 a (6.3)  66.4 a (9.5) 71.7 a (7.4)  78.5 b (3.2) 78.1 b (3.2)  3.77 ab (0.47) 3.60 ab (0.34)  3.69 a* (0.57) 3.35 a (0.35)  12.7 b (13.2) 9.9 a (4.9)  4.9 c (3.4) 2.8 b (2.0)  78.6 b (3.3) 80.1 b (2.0)  3.93 ab (0.36) 3.78 b (0.29)  Sept  July  20.2 b (23.6) 8.5 a (6.1)  3.8 bc (7.3) 1.3 ab (1.3)  79.4 b (1.3) 78.1 b (1.3)  4.15 ab (0.73) 3.91 b (0.45)  Feb  11.3 ab* (11.2) 6.5 a (10.5)  7.6 ab (5.6) 5.6 a (5.6)  1.8 ab (2.2) 1.6 ab (2.2)  76.5 b (2.9) 78.7 b (2.0)  4.08 b (0.27) 4.01 b (0.36)  Nov  14.7* (24.8) 7.4 (7.8)  8.71 (11.0) 6.61 (5.2)  2.76* (3.60) 1.62 (1.9)  75.9 (5.41) 77.3 (3.3)  3.89* (0.49) 3.71 (0.43)  Mean  Seasonal pH, water content and phenolic acid concentration (nanograms/ g o.d. soil), and standard deviation by month and humus-type (non-woody Fm, and woody Hw). Similar letters indicate no significant difference between months within humus-type; * indicates significant difference between humustype within month.  Ferulic  coumaric  Syringic  2.2 ab (2.1) 2.1 a (3.1) 0.6 a (2.1) 0.7 a (1.1)  0.6 a (1.0) 0.5 a (0.4) 0.4 a* (0.4) 0.1 a (0.2)  Hw  Fm  Hw  Hw  Fm  4.9 ab* (5.5) 2.0 a (3.8)  1.6 a (1.8) 0.7 a (0.7)  Fm  Feb  Table 5.1 Continued  136  0.4 a (3.1) 0 (0)  1.7 ab (3.1) 1.0 a (3.1)  6.0 b* (11.8) 1.0 a (1.7) 0.8 a (11.8) 0 (0)  12.9 b (29.3) 13.1 b (13.9)  Sept  11.4 b* (13.6) 0.9 a (1.8)  July  2.1 b* (2.3) 0.4 a (1.1)  2.4 ab* (2.7) 0.8 a (1.3)  1.5 a (2.8) 0.9 a (1.4)  Nov  0.9* (1.9) 0.3 (0.8)  2.6* (5.7) 1.1 (1.9)  7•7* (18.3) 2.8 (6.7)  Mean  Figure 5.2.  0 Jan  2  4  6  8  10  12  14  Mar  Apr  I  May  Jul  Aug  p-Coumaric  p-Hydroxybenzoic  Month  Jun  0  137  Seasonal phenolic acid concentration (ng/g o.d. salal in cutovers.  Syringic  Protocatechuic  Feb  Phenolic acid concentration (ng/g o.d.  Oct  soil)  Nov  Dec  in Hw horizons under  Ferulic  Vanillic  Sep  )  Phenolic Acids in Hw Horizons  Figure 5.3.  0 Jan  10  20  30  40  50  Jul  -*••  138  Oct  Nov  Dec  in Fm horizons under  Ferulic  Vanillic  Sep  )  Seasonal phenolic acid concentration (ng/g o.d. soil) salal in cutovers.  p-Coumaric  Aug  <  Month  Jun  Syringic  May  p-Hydroxybenzoic  Apr  I  Mar  Protocatechuic  Feb  Phenolic acid concentration (ng/g o.d.  Phenolic Acids in Fm Horizons  September.  Since syringic acid occurs in very small quantities  in coniferous lignin, the high concentration presumably must originate from either salal or other angiosperms rooted in the woody layer, or from above—ground leachates of salal. The patterns of phenolic acid concentration in the non—woody horizons  (Table 5.1 and Figure 5.3)  are more complicated than for  woody horizons, differing with season and type of phenolic acid. The phenolic acids vanillic, protocatechuic and ferulic acids, were significantly higher in wet, colder months than in the driest month (September).  For vanillic this was in February,  for  protocatechuic acid in February and May, and for ferulic in November.  For syringic and p—coumaric, the pattern is reversed,  with significantly higher concentrations in drier,  summer months  of July and/or September than the colder, wetter month of February.  If the phenolic acids are released as hydrolysates  from decomposing organic materials then temperature and moisture content would have a great affect on concentration. A short study was done to determine the effects of soil saturation on the release of phenolic acids. filled with non-woody humus,  Leaching tubes were  saturated with distilled water,  then drained to collect soil solution.  and  One treatment was left  drained, while the other was again saturated with distilled water.  Both treatments were drained after 1 week, and the  experiment repeated for a period of 4 weeks.  It was found that  the humus in the saturated columns produced much more phenolics  139  than the drained columns.  These results suggest that the wetter  months should produce more phenolic acids, as is the case for the vanillic, protocatechuic and hydroxybenzoic acids.  However, this  does not explain why the syringic and p—coumaric acids reach their peak concentrations in the drier,  summer months.  These  drier months are coincident with production of flowers and fruits by the salal plant.  It is interesting to note that the syringic  acid concentration in woody humus is as high in September as the non—woody humus.  This may be evidence that salal is directly  producing syringic acid which leaches down into lower humus layers. A seasonal pattern of phenolic acid concentrations was also found by Jalal and Read (1983).  The concentrations cannot be  directly compared because they used an alkali extraction which extracts both H—bonded and free phenolic acids.  Concentrations  were found to be highest in May and through the summer months, and were lowest in winter months.  The seasonal nature of acid  production was attributed to the effects of temperature both directly upon microbial activity and indirectly upon soil water status.  Increasing aeration and temperatures increased fungal  metabolism and growth which were responsible for the production of organic acids from the fatty and phenolic acid rich residues of the healthland raw humus. These results suggest several explanations.  First,  protocatechuic, hydroxybenzoic and vanillic acids are probably  140  readily utilized by microbial populations,  so that during the  colder, wetter months, microbial metabolism has slowed sufficiently to allow accumulation of these acids.  Once  temperature, aeration and microbial metabolism increases, the concentration of these phenolic acids decreases. In contrast, the higher concentrations of syringyl and coumaryl derivatives in the summer months may result from 3 factors: a) phenolic acids associated with salal increase in production when salal is more physiologically active, b) the release of these phenolic acids occurs through microbial decomposition of the phenolic acid rich residues of the salal or other plant litter, or c) these phenolic acids are broken down less easily than the other three and are therefore more readily available and detected.  Some or all of these factors may be  involved. In summary, seasonal concentrations of free phenolic acids under salal suggests that salal may be in part responsible for the production of free phenolic acids, either directly or through microbial metabolism of its phenolic acid rich litter, particularly syringic and coumaric acids.  Whether these  concentrations are sufficient to elicit an allelopathic response by conifers is now examined.  141  3.2.  Effects of Phenolic Acids and Salal Leachates on Conifers 3.2.1  Seed germination  The overall germination performance of the seeds varied greatly with species.  Germination performance was good for the  Sitka spruce, but low for western red cedar, and very poor for western hemlock.  This probably occurred because of the  differences in age of the seeds, the Sitka spruce being the youngest and the western hemlock the oldest.  Comparisons can  nevertheless be made within species between treatments and pretreatments. The effect of the solutions varied with species and pretreatment.  Germination capacity (GC) was not significantly  different by treatment for those seeds which were unstratified, regardless of species, but stratification resulted in treatments having significant effects within species  (Table 5.2).  For Sitka  spruce, the stratification treatment resulted in significantly greater GC than the unstratified treatment.  This is consistent  with the need for stratification treatment in Sitka spruce.  For  seeds given the stratified pretreatment, the phenolic solution resulted in significantly lower GC than the control and soil solutions,  and the salal treatment was significantly lower than  all other treatments. For western red cedar, the effect of pretreatment gave no significant difference in GC between the control and soil solution treatments, but gave significantly lower GC for the 142  Table 5.2:  Germination indices by species, pretreatment and treatment. Treatments with similar lower case letters are not significantly different between pretreatments; treatments with similar upper case letters are not significantly different within pretreatments.  a) R50 Species  Pretreatment  Ss  Strat Unstrat  Wh  Strat Unstrat  Wrc  Strat Unstrat  b)  Phenolic  Treatment Soil Salal  Control  9.3 dB 12.8 cB  8.9 deC 13.5 bB  8.7 eC 12.9 cB  0 0  0 0  12.6 cB 14.0 bcA  13.4 bA 15.9 aA 0 0  9.7 dC 12.6 cB  18.1 aA 14.6 bA  0 0 10.2 dC 12.4 cB  Germination Capacity  Species  Pretreatment  Treatment Soil Salal  Phenolic  Control  Ss  Strat Unstrat  92.3 aA 90.0 abA  95.5 aA 84.8 bA  94.3 aA 85.0 bA  94.0 aA 90.0 abA  Wh  Strat Unstrat  27.8 abA 24.5 abA  33.3 aA 26.0 abA  19.8 bB 27.0 abA  26.5 abA 30.0 abA  Wrc  Strat Unstrat  65.0 bB 74.5 aA  76.5 aA 71.3 abA  55.8 cC 70.5 abA  77.3 aA 75.8 aA  c)  Germination Value  Species  Pretreatment  Treatment Soil Salal  Phenolic Ss  Strat Unstrat  Wh  Strat Unstrat  Wrc  Strat Unstrat  31.9 bB 21.6 dA  36.2 aA 17.6 eB  0.7 abAB 0.5 abA 13.1 bB 14.9 bAB  1.2 aA 0.6 abA 22.1 aA 14.8 bAB  143  24.9 cC 16.7 eB 0.3 bB 0.7 abA 7.6 cC 12.8 bB  Control 35.9 aA 21.0 dA 0.7 abAB 0.8 abA 21.1 aA 16.8 bA  stratified pretreatment in the phenolic and salal solution treatments.  This is presumably the effect of stratification,  which actually stresses the seeds.  Those seeds that were  stressed by stratification, were then further stressed by the phenolic and salal solution,  to a point where they could not  germinate. For western hemlock, there was no difference in GC with pretreatment.  Those seeds given a stratification treatment, the  salal treatment was the only treatment with a CC significantly lower than the control. The R 50 value, which is a measure of the rate of germination, could not be calculated for western hemlock because the germination capacity was so low that 50% germination was not reached.  For Sitka spruce and western red cedar,  stratification  50 than no pretreatment for all resulted in a significantly lower R solution treatments.  The R 50 was not significantly different  between the soil and control treatments regardless of pretreatment, and the soil treatment was slightly, but not significantly lower for the stratified pretreatment. spruce,  For Sitka  the phenolic treatment was significantly lower than the  soil and control treatments for stratified, but not for 50 than unstratified. The salal solution had significantly lower R all other treatments regardless of pretreatment.  For western red  50 than cedar, the phenolic solution had a significantly lower R control and soil solutions,  for both pretreatments,  144  and the salal  solution was significantly lower than the phenolic solution for the stratified treatment only. The GV,  is a factor which combines rate of germination with  germination capacity.  The GV for the soil solution was higher  than that of the control for the stratified pretreatment, regardless of species, although the differences were not always significant.  This indicates that the soil solution had some  benefit on germination.  For western hemlock, there was no  significant difference between pretreatment, and no significant differences between treatment solutions for unstratified seeds only.  However,  for the stratified seeds, the GV was  significantly different between the soil solution and the salal solutions only.  For stratified Sitka spruce and western red  cedar, the phenolic solution had a lower GV than the control and soil solutions,  and the salal solution was significantly lower  than the phenolic solution. In summary,  stratification tended to give faster germination  rates for the species tested, but resulted in lower germination capacities for western red cedar, higher germination capacities for Sitka spruce, and no difference in western hemlock.  The  stratified seeds were more susceptible to the treatment effects. None of the germination indices were significantly different between soil and control treatments; for this reason and because of the difficulties involved in obtaining adequate quantities of soil solution, the soil solution treatment was not continued in  145  subsequent bioassays.  Western red cedar was always significantly  more affected by the salal solution, than by the phenolic solution, but the phenolic solution was still significantly different than the control solutions.  For Sitka spruce, this  same trend was apparent for the R 50 and the GV indices.  For  western hemlock, the salal treatment gave the lowest GC and GV, although they were not significantly different from the control.  3.2.2 seedling growth  Results of the seedling biomass study are presented in Table 5.3.  There were no significant differences in root biomass, but  shoot biomass for salal treatment was significantly lower than the control treatment for all species.  Shoot biomass for the  phenolic treatment was significantly lower than for the control treatment for Sitka spruce only,  and was significantly greater  than the salal treatment for Sitka spruce and western red cedar. Total biomass for Sitka spruce was significantly lower than the control for both the phenolic and salal treatments.  For western  hemlock, biomass of seedlings given the salal leachate solution was significantly lower than the control, but the phenolic acid treatment was not significantly different from control or salal treatments.  Biomass of western red cedar given the salal  treatment was significantly lower than both control and phenolic treatments. It is important to consider that the various treatments had  146  different pH values: control, pH 6; phenolic solution, pH 5; and leachate solution, pH 4.  The effect of the solution pH cannot be  separated from the effects of the compounds within the solution. The natural soil solution pH was found to be 3.9 for Fm horizons and 3.7 for Hw horizons, similar to natural pH.  so that of the leachate solution is most This means though that the control  solution which provides a more favourable pH, may be biasing the results.  However, these differences in pH may reflect natural  conditions; rainfall falling directly onto forest soils would be close to neutrality; rainfall passing over salal biomass before reaching the soil,  could contain leachates at a much lower pH;  and soil solution pH is near 4.  However, these effects would be  important and must be considered when discussing the various treatment effects. Also, the treatment effects may be exacerbated because as the rooting medium dries, the solutions would concentrate, thereby leading to concentrations of potential toxins that are much higher than used at the time of watering.  A similar effect  could be seen under field conditions as soils dry out in the summer,  but the drying would be much more gradual.  3.2.3  seedling root bioassay  Short term uptake of inorganic phosphorus  (Pi)  in distilled  water by roots of seedlings which had been germinated and watered with solutions of phenolic acids or salal berries and flowers is  147  Table 5.3:  Species  Seedling shoot, root and total biomass (mg) and uptake of inorganic P (nanoMoles per g oven—dry root) by species and treatment. Treatments followed by the same letter are not significantly different within species. Treatment Shoot (mg)  Ss  Wh  Wrc  Biomass Root (mg)  Pi Uptake Total (mg)  (nNol/ci)  Control  22 c  10 a  31 b  15.93a  Salal  14 a  9 a  23 a  19.88a  Phenolic  18 b  8 a  25 a  17.77a  Control  24 b  7 a  31 b  6.21a  Salal  11 a  6 a  17 a  6.84a  Phenolic  15 ab  7 a  22 ab  7.37a  Control  13 b  6 a  19 b  4.44b  8 a  5 a  13 a  5.39b  13 b  5 a  18 b  1.50a  Salal Phenolic  148  shown in Table 5.3.  There was no significant difference between  treatments for Sitka spruce or western hemlock, but the phenolic treatment resulted in significantly lower uptake of Pi for western red cedar.  The lack of significance for the phenolic  treatment on Pi uptake for Sitka spruce and western hemlock is consistent with studies by Glass and Dunlop (1974)  and Harper and  Balke (1981) who found that the inhibition of Pi or K uptake by phenolic acids was almost immediately reversed on removal of the phenolic solution. Uptake of Pi was higher  (but not significantly so),  salal and phenolic treatments than for the controls.  for the  This can be  explained by the widely observed phenomenon that plants have the capacity to increase transport capacity in response to low nutrient availability (Glass,  1989).  This suggests that the Pi  uptake of the seedlings given the phenolic acid or salal leachate solutions were below the optimum level such that when the seedlings were removed from that environment, uptake increased immediately.  Uptake may be below the optimum because the phenol  and salal treatment are either reducing ion uptake directly, or they inhibit mobilization of the Pi in the soil, or that the pH differences are affecting uptake. This is examined further in the next section. The significant reduction in Pi uptake for the phenolic treatment in western red cedar suggests that root damage may have occurred.  Harper and Balke  (1981)  149  found that absorption of  salicylic acid at a low pH of 4.5 caused sufficient membrane damage to allow leakage of ions and organic solutes out of the root cells of oats.  The pH of the greenhouse growing medium in  this study was about 5.  That only western red cedar was affected  may reflect the fact that species differ in response to phenolic acid additions  (Harper and Balke,  1981).  This suggests that  effects of salal leachates and phenolic acids at maximum soil solution concentrations found, do not have irreversible effects on the Pi uptake capacity in Sitka spruce or western hemlock roots, but the phenolic acid concentration may impair root functioning in western red cedar. Some problems with the technique of McDonald used for this bioassay became apparent.  (1992)  Adequate aeration,  important for oxidative respiration of roots, was not maintained during the immersion of the roots in the solutions, as is normally done Furthermore,  (Harper and Balke,  1981; Glass,  1974).  the roots were washed in distilled water rather than  in a similar unlabelled solution, which would tend to cause an immediate change in ion uptake by passive diffusion.  These  problems are not addressed in the published methods, but should be considered in future studies.  3.2.4 Mature Root Bioassay  Uptake of inorganic phosphorus  (Pi)  various solutions is shown in Table 5.4.  150  by mature roots in the Again,  it is important  to consider the pH effects of the treatments, as well as the solutions themselves.  For western red cedar the salal leachate  solution, which had the lowest pH, had the greatest effect by causing a significant reduction in Pi uptake to about 15% of control levels; the phenolic acid solution caused a significant reduction in uptake to about 36% of control levels.  For western  hemlock, the salal treatment caused a significant reduction in Pi uptake to about 9% of control level; the phenol treatment caused a reduction in uptake to 69% of control levels, but this was not significantly different from the control. The fact that western red cedar is more strongly affected by the treatments than western hemlock is consistent with the earlier findings for roots of western red cedar seedlings.  The  greater reduction of uptake by the salal leachate solution over that of the phenolic acid solution may result not only from compounds in the solution, but also from the greater acidity (pH 4 versus pH 5 of the phenolic solution and pH 6 of the distilled water).  Ideally,  the solutions.  the pH should have been kept constant between  However, the lower acidity is not different from  that of the pH of soil solution occurring naturally, varying between 3.5 and 4.1.  In fact, the effect of the phenolic  treatment may be even greater under natural soil pH conditions. Harper and Balke  (1981)  examined the interactions of the effect  on K uptake of phenolic concentration and pH and found that much lower phenolic concentrations were required to produce the same  151  Table 5.4: Root biomass (mg) and uptake of inorganic phosphorus (Pi) in nanoMoles per oven—dry g of mature roots. Treatments with the same letters are not significantly different within species.  Species  Treatment  Root Biomass (mci)  Pi Uptake (nMol Pi/ci)  Relative Uptake (% Control)  Wrc  Control  33 a  39.29 c  100.0  Salal  25 b  5.82 a  14.8  Phenolic  33 a  14.00 b  35.6  Control  24 a  30.00 b  100.0  Salal  28 ab  2.80 a  9.3  Phenolic  31 b  20.55 b  68.5  Wh  152  effect at low pH as that of higher concentrations of phenolics at higher pH.  4.0  SUMMARY The concentration of free phenolic acids under salal were  found to vary with horizon and season.  The yearly mean  concentrations of free phenolic acids in soil solution under vigorously growing salal in young conifer plantation, was significantly higher in non-woody humus materials than woody humus for all phenolic acids tested except p-hydroxybenzoic acid which did not differ significantly.  Concentrations of vanillic,  protocatechuic and p—hydroxybenzoic acids were significantly higher in colder, wetter months, while that of syringic and p coumaric were higher in warmer, drier months.  Syringic acid  concentration was found to be as high in the woody humus materials as in the non—woody humus in the driest month.  This  suggests that syringyl and coumaryl units originating from salal are at their highest concentrations when the young conifer trees are most drought stressed. The effect of the phenolic acids at soil solution concentrations and of salal leachates on conifers was then examined.  It is important to remember that the solutions were  not buffered,  so the overall treatment effects includes both the  pH and allelochemical effects.  Germination of stratified western  red cedar and Sitka spruce seeds was significantly affected by  153  both the phenolic solution and the salal leachates, with the salal treatment having significantly more effect than the phenolic treatment.  Western hemlock did not appear to be  significantly affected, but this may reflect poor seed stock more than actual treatment affects. The effects of phenolic acids at pH 5 and salal leachates at pH 4 were tested on total biomass of seedlings.  After twelve  weeks growth, there was no significant difference in root biomass between treatments for any of the species tested.  However, total  biomass was significantly lower for the salal leachate treatment than for control treatments for all species tested.  Total  biomass was lower for the phenolics but significantly different from the controls only for Sitka spruce.  Longer term treatments  could produce more significant differences. The 32 P root bioassay of the mature conifer roots indicates that short-term uptake of Pi is affected by the nanomole concentration of phenolic acids found in soil solution at pH 5. For western red cedar, uptake was only was 36% of the control solution which was highly significant.  For western hemlock,  uptake was 69% of the control, but this was not highly significant.  The effect of the salal leachates at pH 4 is even  more dramatic, with only 15% and 9% of control uptake in western red cedar and western hemlock respectively. The effect of the treatments over a twelve week period on root uptake in distilled water were not significantly different  154  for western hemlock or Sitka spruce.  This reversibility of the  phenolic effect (as compared with significant short term effects as tested on the mature roots)  is in agreement with Glass and  Dunlop (1974), who found that the effect of the phenolics is most likely at the root membrane,  since recovery was almost immediate  when roots were transferred to phenolic—free solutions.  The  significantly lower uptake in the phenolic treatment of western red cedar indicates that irreversible root dysfunctioning may have occurred.  Root damage by phenolic acids at low pH was also  found by Harper and Balke (1981).  The inorganic phosphorus  uptake in the phenolic and salal leachate treatments tended to be higher than the controls, which may indicate that the seedlings were deficient in Pi relative to the controls.  5.0  CONCLUSION 5.1 Seasonal Phenolic Acid Concentrations Under Salal Hypothesis:  That the concentration of free phenolic acids  found under salal in plantations will vary with season and will be highest during the summer months when salal is most physiologically active. Conclusion: Accept that concentrations vary with season,  and that syringic and p—coumaric acids are  highest during the summer months.  155  5.2 Effects of Phenolics on Conifers  Hypothesis:  That solutions using the maximum concentrations  of free phenolic acids found in soils under salal and of leachates from the flowers and berries of salal,  will cause  reduced seed germination, biomass growth, and 32 P uptake in roots of Sitka spruce, western hemlock and western red cedar. Conclusion: Accept  156  CHAPTER VI  Overall Discussion  1.0  INTRODUCTION The causes for growth—check of regenerating conifers on the  CH phase, but not the HA, on Vancouver Island involves many interacting factors.  The differences in productivity begin long  before the stands are cut,  so it is important to understand the  basic differences between CH and HA phases. The terms CH and HA phases were first used by Lewis to describe site differences within the Thula plicata heterophylla “salal  —  -  moss”  Gaultheria shallon ecosystem  of  -  -  (1982) Tsuga  Rhytidiadeiphus loreus or  the windward, submontane, wetter  variant of the wet subzone (CWHvm)  of the Coastal Western Hemlock  biogeoclimatic zone on northern Vancouver Island.  This single  ecosystem association was subdivided based on what appeared to be successional differences.  The cedar—hemlock (CH) phase, the  157  climatic climax community, consisted of old (perhaps 1000 years), somewhat open, western red cedar and western hemlock stands with a minor component of amabalis fir and a dense understorey of salal.  The hemlock-amabalis fir (HA)  phase,  a seral stage  occurring on sites with a history of soil disturbance, consisted of young (about 80 years)  even—aged, densely stocked, western  hemlock and amabalis fir, with an understorey largely composed of mosses and sparse shrub and herb cover.  The occurrence of  communities intermediate between the climatic climax CII phase and the seral HA phase lead Lewis  (1982)  to state that the HA phase  had the potential to develop into the CH phase, given a sufficiently long period of time without soil disturbance. In this study, the CH and HA phases were found to occur side by side, but the HA phase occurred on drier ridgetops, while the CH phase were directly adjacent on lower, wetter slopes. difference alone profoundly affects the site history, structure,  This  stand  and corresponding differences in physical and chemical  soil properties between CII and HA phases.  The higher topographic  position of the HA phase, makes the site more susceptible to windthrow.  The most recent catastrophic winds occurred in 1908,  and most of the HA stands examined in this study originated from this single wind event.  The dense,  even—aged structure renders  the stands susceptible to subsequent wind—throw events, and the process of stand renewal to even—aged conditions is repeated. these stands,  salal is rarely seen and other understorey  vegetation is sparse, presumably because the dense structure of  158  In  the stand prevents light penetration to the forest floor.  In  contrast, the undisturbed CH stands are relatively open and uneven—aged, which encourages windfirmness (Oliver and Larsen, 1991). Windthrow disturbances were found to be very important in causing differences in pedogenic processes.  Windthrown trees  tend to create pits and mounds and mix the soil horizons  (Bowers,  The mixing of the soil by windthrown trees has been found  1987).  to contribute to improved soil productivity by favouring organic matter decomposition, releasing tied—up nutrients, soil,  aerating the  breaking hardpans and reversing the process of  podsolization (Bowers,  1987).  The differences in pedogenic  processes resulting from windthrow are seen in soils from CH and HA phases. The forest floor humus profiles were found to consist of six major humus horizons, three of woody origin and three of non— woody origin, varying in degree of humification. horizons Fw,  (given the suffix w)  The woody  included the Fw, Hrw and Hw.  The  consisted of over 90% poorly decomposed wood in which the  woody structure held when rubbed between the fingers.  The Hrw or  “residuic” woody H horizon consisted largely of rooting wood, but the amorphous component was greater than 20%,  and the woody  structure failed when rubbed between the fingers. humified woody horizon,  The Hw, or  consisted of less than 20% woody and over  80% amorphous materials with a crumbly, greasy structure. non-woody horizons included the Fm, Hh and Hhi. 159  The Fm  The  (mycogenous F horizon)  consisted of a mix of plant,  fungi and  amorphous materials with a compact and matted structure. (humified H horizon)  The Hw  consisted of greater than 80% amorphous  materials and has a massive, block and greasy structure.  The Hhi  (humified intrusive horizon) was very black and greasy consisting of over 95% amorphous materials and tended to occur only at the forest floor—mineral soil interface. The classification of humus horizons in this study had excellent correspondence with the spectra of ‘ C NMR. 3  Woody  horizons were dominated by signals from lignin, but with increasing decomposition, the relative proportion of lignin decreased, while aliphatics and carbohydrates increased, presumably from microbial.  The non—woody humus types were  typical of forest litter layers, which are dominated by signals in the carbohydrate region.  Increasing decomposition resulted in  decreasing carbohydrates and increasing aliphatics and carboxyl. Although all six humus horizons were found to occur on both CH and HA sites,  their relative abundance was found to vary.  This variation was found to be associated with site history.  The  windthrow process was responsible for the deposition of a large amount of woody material on HA sites. horizons  Poorly decomposed woody  (Fw and Hrw) were found to occur on 68% of HA plots, but  only 38% of CH plots.  Well decomposed 11w horizons were found on  56% and 38% of HA and CH plots respectively.  Where they did  occur, there was not significant difference in depth of woody  160  horizons between CH and HA.  The non—woody horizons Fm, Hh and  Hhi were deeper and occurred more often on the CH plots. Soil profiles along a trench through one HA site consisted largely of lignomors  (41%) with greater than 35% woody debris.  Within the lignomors was an equal proportion of young Hrw)  and old  (Hw)  (Fw and  horizons indicating that the windthrow process  has been repetitive.  HA phase soils consisted of a large  proportion of hemimors  (37%), characterised by thin Fm horizons  over windthrow—disturbed, mixed organic—mineral mounds.  Only 22%  of the trench consisted of humimors, characteristic of mature, undisturbed profiles. and well—drained.  Mineral soils were friable, well—aerated  Pans were found under the humimors, but these  tended to be thin and discontinuous. In contrast,  soil profiles along a trench through a CH stand  consisted of a relatively large proportion of humimors  (57%),  with characteristic well developed H horizons reflecting maturity and lack of disturbance.  Lignomors comprised 17% of the soils  with a larger proportion of older (Hw) horizons, relatively old woody inputs.  indicating  Hydromors, which develop under the  influence of excessive moisture on poorly drained soils, were also common  (24%).  which develop,  Hydromors are poorly aerated humus forms  in part, under hydrolysis and periodic anaerobic  fermentation, which results from standing water near the mineral soil surface.  The mineral soils of the CH trench tended to be  compact, with continuous, thin pans over which humus—enriched Bf layers were found,  reflecting a lack of disturbance. 161  The inherently different site histories and their associated pedogenic processes make the CH and HA sites quite different in terms of potential productivity. from water,  Root restricting layers arising  compacted soils or pans tended to be much more  frequent on CH than HA sites  (98% vs.  70% respectively) with the  result that the CH sites had shallower mineral soil rooting depths than HA (21 vs.  Also, the presence  29 cm respectively).  of gleyed horizons occurred much more frequently on CH than HA (30% vs.  12% respectively).  The complexities of the origin (woody vs. non-woody), of decomposition,  state  and depths of forest floor profiles made bulk  sampling for nutrient analysis unfeasible.  Therefore,  each of  the six horizon identified were sampled for chemical analysis. Nutrient concentrations were found to vary between horizons.  Not  surprisingly, the woody humus was found to be significantly lower than non-woody humus for pH, total N and S, N,  S and P,  and total inorganic K and Mn.  C/N ratio,  available  Nutrient  concentrations tended to increase with increasing decomposition of woody horizons from Fw to Hrw to Hw. found to have abundant fine roots,  The Hw horizons were  and were thus the most  biologically important of the woody horizons.  Nutrient  concentrations within the Hw horizon were found to differ between CH and HA sites; the pH, moisture content and C/N ratio of those from the CH were higher than those from the HA, while total N and S,  and available N and P were significantly higher for the HA  compared to the CH.  The higher pH of the CH probably reflects 162  the higher pH of litter associated with CH sites, red cedar and salal.  such as western  The higher moisture content is not  surprising, giving the earlier findings that the CH tends to occur on wetter sites.  But the nutrient concentration  differences seems to indicate that the CH site is either decomposing at a slower rate than the HA, or that decomposition is less complete.  Again, this may be a function of the wetter CH  site, or to differences in litter inputs. For non—woody horizons, the Fm horizon, which is the most biologically active horizon in terms of root abundance, was found to be significantly higher than other non-woody horizons in available P and exchangeable K and Mn.  There were no significant  differences in nutrient concentrations between Fm and Hh horizons.  The Hhi horizon had significantly higher ash contents,  and total C, N and S.  The high ash content reflects the fact  that the Hhi is located at the humus-mineral soil interface.  The  higher concentration of total C, N and S probably reflects the advanced state of humification.  The Hhi horizon was felt to be  the least biologically active of all humus horizon. For the Fm horizon,  samples from the CH sites were found to  be significantly higher than those from the HA for pH, moisture content,  available 5,  and exchangeable Ca, K, and Mn; this most  likely reflects differences in litter input between the sites, and to the wetter conditions of the CH.  For the Hhi horizon,  samples from the CH sites were found to be significantly higher than for the HA for pH and water content, but available P was 163  higher for HA than for CH. Lipids, which arise from undecomposed plant material and from the bodies of microfaunal organisms, ranged between 1 and 9%.  Not surprisingly, non-woody horizons tended to have higher  lipid concentrations than woody horizons, and concentrations increased with increasing decomposition.  The CH sites had  consistently higher and more variable lipid concentrations than HA sites.  Similarly, C’ 3 NNR indicated that the more decomposed  Hrw, Hw and Hhi horizons from the CH site had a greater relative percentage of C in the aliphatic region than did similar horizons from the HA.  This may occur for several reasons: the  concentrations of lipids originating from salal or western red cedar litter may be higher than that of litter found on the HA; the wetter site conditions of the CH may inhibit the breakdown of lipids by microorganisms leading to an accumulation; or microorganisms synthesize a larger quantity of lipids on CH sites. Total and labile polysaccharides and cellulose were found to decrease with increasing decomposition and were all found to be higher for CH horizons than for HA.  The same trends for  cellulose was also found using C’ 3 NMR.  This suggest,  as with the  lipid and nutrient concentration differences between sites, that the CH site seems to be decomposing either at a slower rate than the HA,  or that decomposition is less complete.  Tannins were identified in the Fm layers of both CH and HA sites using C’ 3 NMR, but intensity was greater on samples from the 164  CH phase.  Tannins were also identified in the leaves, roots,  flowers and litter of salal using 3 ‘ NMR. C  Furthermore, the juice  of salal berries were found to contain large quantities of Both the flowers and berries may thus be a source of  tannins.  readily leachable tannins, particularly in cutovers, while litter inputs from leaves and roots provide a source of tannins through decomposition.  The tannin from salal leaves was thought to be a  either a procyanidin or a procyanidin and prodeiphinidin mix. Tannins are known to reduce the biodegradability and humification of organic matter by three processes: the production of protein—tannin complexes which are much more resistant to microbial decomposition than unaltered proteins; the permeation and coating of nonproteins such as cellulose and hemicellulose by the protein—tannin complexes, giving them considerable resistance to microbial attack; and by the inactivation of enzymes important in the process of decomposition. to lignin C horizons,  (as found by C’ 3 NMR)  The ratio of total carbohydrate tended to be higher in CH humus  indicating that the carbohydrates may be more resistant  to decomposition, possibly as a result of the tannins.  Thus  lower concentrations of available nutrients, and higher concentrations of lipids, polysaccharides, humus horizons may be occurring,  and cellulose in CH  in part, because of the effects  of tannins arising from the presence of salal on CH, but not HA sites.  Other associated factors which could be affecting rate of  decomposition are the wetter site conditions of the CH phase, and  165  also the presence of fatty acids, which were not examined in this study. A comparison of bound phenolic acid concentrations between humus horizons indicated that woody horizons tended to be higher in vanillic acid while the non-woody horizons tended to be higher in protocatechuic, p—coumaric and ferulic acids.  The Fm horizon,  with the greatest proportion of angiosperm inputs, had the highest concentrations of protocatechuic acid, degradation product of angiosperm lignin), ferulic acids  syringic acid (a  and p—coumaric and  (thought to originate from the cutin of leaf and  other plant tissues and from the suberin of roots); the Fw, which is pure coniferous wood, had the highest concentration of vanillic acid (a degradation product of coniferous lignin); the Hhi, was found to be highest in p-hydroxybenzoic acid. Interestingly, the Fm, which is the most biologically active horizon, was found to have the highest overall concentration of phenolic acids, while the Hhi, which would be expected to accumulate the mobile organic acids from overlying horizons, was found to have the second highest overall concentrations of phenolic acids. When the CH site, with characteristically abundant salal was compared with HA sites, with relatively sparse understorey vegetation,  the CH site was found to be significantly higher in  syringic, p—coumaric and ferulic acids.  The concentration of p—  hydroxybenzoic acid, originating from suberin of roots, was not significantly different between sites, but tended to be higher on 166  CH sites than on HA. The concentration of free phenolic acids in soil solution under salal in clearcuts was examined.  Average concentrations  over five sampling periods in one year, were found to be in the range of nanograms per gram of oven—dry soil.  Woody horizons had  significantly lower phenolic acid concentrations than non—woody horizons.  Concentrations in woody horizons did not vary  significantly over the year, with the exception of syringic acid which peaked very sharply in September to concentrations as high as that found in non—woody horizons.  Since syringic acid occurs  in very small quantities in coniferous lignin, the high concentrations could originate from either salal rooted in the woody layer,  or from above—ground leachates of salal.  The concentrations of phenolic acids in the non—woody horizons varied significantly over the course of one year.  The  wetter winter months had the highest concentrations of vanillic, protocatechuic,  and ferulic acids.  This probably occurred  because microbial metabolism had slowed sufficiently to allow accumulation of these acids.  Once temperature, aeration, and  microbial metabolism increased, concentrations of phenolic acids decreased.  In contrast, the drier summer months had the highest  concentrations of syringic and p—coumaric acids. from several possible processes: a)  This may result  these phenolic acids are  associated with salal and increase in production when salal is most physiologically active as, flowers or berries; b)  for example,  leaching from the  the release of these phenolic acids  167  through decomposition of salal litter exceeds the utilization by microbes in the summer,  leading to higher concentrations, and c)  these phenolic acids are less transient than the other phenolic acids.  It is unclear which process would be more important.  The low concentration of these phenolic acids made a bioassay important.  The effects of the maximum concentrations of  phenolic acids found in the field, and of a 5% solution of salal berry and flower leachates were examined on conifer seed germination, seedling growth and root phosphorus uptake.  It is  important to remember that the solutions were not buffered, so the overall treatment effects include both the pH and allelochemical effects. For Sitka spruce and western red cedar, the germination value, which combines germination rate and capacity, was significantly lowest for the salal leachate solution, while the phenolic solution was significantly lower than the control solution.  The results for western hemlock germination value were  not significant, but this may reflect poor seed source, since the seeds had been stored for over ten years.  The lower germination  values could be significant for naturally regenerating seedlings in clearcuts. Biomass of seedlings were affected by weekly watering with the treatment solutions.  Root biomass of Sitka spruce and  western red cedar seedlings given the salal leachate or phenolic acid solution were smaller, but not significantly, than those given the control solutions.  For western hemlock seedlings, root 168  biomass of seedlings given the salal leachate treatment were smaller, but not significantly, than those given the control solution. The salal leachate treatment resulted in shoot biomass of all seedlings that was significantly smaller than those given the control treatment; the phenolic treatment solution resulted in shoot biomass of Sitka spruce that was significantly smaller than those seedlings given the control treatment.  The results  indicate that seedling biomass can be significantly affected by the salal leachate or soil phenolic acid solution.  If field  conditions were to result in the same trend towards smaller seedling biomass, this could be significant to the overall productivity of the trees.  Whether the effect of the phenolic  acids and salal leachate treatment is direct or indirect was examined using radioactive phosphorus uptake in roots. When root samples taken from mature trees were placed into the treatment solutions augmented with a 32 P—labelled phosphorus solution  (unbuffered), phosphorus uptake was significantly lower  in treatment solutions than for controls. The phenolic acid solution reduced uptake to 36% and 69% of that of controls in western red cedar and western hemlock respectively.  The tannin  solution had a even more pronounced effect, reducing phosphorus uptake to 15% and 9% of that of controls in western red cedar and western hemlock respectively.  These short term effects appear to  be reversible over the long term, western hemlock.  at least for Sitka spruce and  When the roots of seedlings which had been 169  watered with the treatment solution were placed into a labelled phosphorus solution without the treatments, the shortterm uptake was significantly lower for the phenolic acid treatment in western red cedar only.  The total inorganic P  uptake in Sitka spruce and western hemlock were actually higher for the treatments than for the controls significantly so).  (although not  The higher total inorganic uptake in the  salal and phenolic acid treatments can be explained by the widely observed phenomenon that plants have the capacity to increase transport capacity in response to low nutrient’availability. This suggests that the seedlings were deficient in P and, given the new conditions of higher P concentrations, uptake increased. The phosphorus deficiency may have resulted from reduced ion uptake either directly at the root by phenolic acids or tannin leachates,  (as was found in the mature root bioassay), or  indirectly by inhibition of P mineralization in soils containing tannins  (as was found in CH humus).  The significant reduction in inorganic P uptake for the phenolic treatment in western red cedar suggests that root damage may have occurred in the long term.  This may be because the  phenolic concentration would increase as soils dried out between watering. The overall hypothesis of this study, that there are differences in chemical characteristics of similar humus horizons between CH and HA sites,  and that these differences may be the  cause of the poor productivity of conifer regeneration on 170  clearcut CH sites is therefore accepted. The implications of this study for the management of CH phase cutovers are not straightforward.  If the windthrow process  is important in rejuvenating the site, then scarification should replicate the process by breaking up hardpans, aeration, horizons.  increasing soil  and improving soil fertility by mixing mineral and soil A trial at Port McNeill is presently underway which  examines this treatment. Another alternative is to remove salal from the site, and to avoid situations in which salal is allowed to reestablish in open—grown conditions.  In practice, however, this is difficult  or almost impossible to do.  Slashburning, physical site  preparation, prompt and dense conifer regeneration, and tree fertilization to speed up crown closure are methods presently used to eliminate salal on CH sites. eradicate salal using herbicides.  Some work has been done to  Field research trials using  Garlon with a diesel oil delivery has been shown to be reasonably effective.  It is unlikely that this technique will be widely  used in commercial forestry.  Some evidence also exists that a  solution of calcium nitrate at a rate of 1.13 micromoles per gram of soil, may reduce the buildup and toxicity of phenolic compounds in agricultural soils  (Farquharson  j.,  1990).  This  may occur because phenolic acid toxicity is greatest in its undissociated form at low pHs of 3 to 4,  or that calcium can aid  in the adsorption of phenolics onto clay or humus particles rendering them inactive (Rice,  1984).  171  However,  in a forestry  situation, rates of fertilization needed to get the necessary increase in pH could be excessive. One possible treatment, which has not been tried, would be to use red alder (Alnus rubra), a nitrogen—fixing, tree,  indigenous  as a nurse crop in plantation following scarification.  This would improve the nitrogen balance of the site, add a large annual source of readily decomposable litter, rapid shading of salal understorey.  and contribute to  No trials have been  initiated to date. Fertilization with nitrogen and phosphorus is the best known silvicultural tool to relieve the growth-check of conifer regeneration, even though salal responds well to fertilization with increased above— and below—ground vigour.  Any toxic effects  of phenolic compounds are not as great following fertilization (Stowe and Osborne,  1980)  and fertilization may also cause a  priming effect, resulting in an increase in nitrogen mineralization and decomposition rates of humus.  Furthermore,  applications of fertilizer that result in an increase in the density of the conifer crown canopy cause a decline in the vigour and cover of the salal understorey.  172  CHAPTER VII  Summary and Conclusion  1.0  SUMMARY  1.  Differences between the CH and HA phases begin with differences in topographic position.  The HA phase,  which tends to occur on drier ridgetops,  is more  susceptible to windthrow events than the lower, wetter topographic position of the CH phase.  The resulting  differences in stand structure are such that HA stands are dense, young, ancient  even—aged,  while those of the CH are  (possibly 1000 years since last disturbance)  fairly open stands with abundant understorey vegetation dominated by salal.  2.  Six distinct humus horizons were found to occur commonly on the CH and HA sites.  173  They can be  distinguished on the basis of origin (woody vs. nonwoody)  and on the degree of decomposition (fermented to  well—humified).  Woody horizons were found to be lower  in nutrients, total and labile polysaccharides and bound phenolic acids than were non—woody horizons. Lipids were also lower in woody horizons,  except that  the Hw horizon had very high concentrations.  3.  CII sites were found to have a higher proportion of well humified woody and non—woody horizons, reflecting ecosystem maturity and a lack of disturbance.  The HA  sites were found to have a greater proportion of so— called residuic” woody horizons, more friable mineral soils, and less root restricting layers than the CII sites,  4.  indicative of repetetive windthrow events.  Although not consistently significant, the following trends in nutrient concentrations were apparent: CH humus horizons differed from HA humus horizons in that they tended to have higher concentrations of K, Ca, Mn and available S,  lipids, and total and labile  polysaccharides, as well as a higher pH.  Some of the  differences can be attributed to the greater inputs of litter from western red cedar and salal on CII sites. The HA humus horizons were found to be higher in available N and P and tended to have a lower C/N ratio 174  for the more well decomposed horizons.  These findings  suggest that for the same humus horizons,  those from  the HA sites are better able to achieve a more advanced state of decomposition than the CH. conditions of the CH phase,  The wetter site  or the presence of tannins  from salal are possible reasons why.  5.  Carbon-l3 Nuclear Magnetic Resonance confirmed the morphological distinction between the six humus types and that these humus horizons are similar between sites.  Woody horizons were dominated by signals from  lignin, but with increasing decomposition, the relative proportion of lignin decreased, while aliphatics and carbohydrates increased, presumably from fungal sources.  There were no apparent differences between CH  and HA sites for woody horizons.  Non—woody horizons  were dominated by signals from the carbohydrates but with increasing decomposition, the relative proportion of carbohydrates decreased and aliphatics and carboxyl increased.  The ratio of carbohydrates to lignin C  tended to be higher for CH horizons,  indicating that  the carbohydrates may be more resistant to decomposition than for the HA horizons.  6.  Tannin signals were found in the Fm horizons of both CH and HA, but the intensity was greater on the CH 175  samples. root,  Tannin signals were very strong in salal  leaf,  flower,  litter and berries.  The tannin is  a proanthocyanidin, and has been tentatively identified as a mix of procyanidins and prodeiphinidins.  7.  Concentrations of free phenolic acids under salal on cutovers were found to vary with season. Concentrations were higher in non—woody horizons than in woody horizons.  Concentrations of vanillic,  protocatechuic and p-hydroxybenzoic acids originating from conifers, were significantly higher in the colder, wetter months.  Concentrations of syringic and p—  coumaric acids were significantly higher in warmer, drier months.  In fact, the concentrations of syringic  acid was found to be as high in the woody humus as in the non-woody in the driest month.  Syringic acid is  not a degradation product of coniferous lignin, and high concentrations are coincident with physiological activity of salal, particularly flower and fruit production in the summer months.  8.  Phenolic acid solutions at field concentrations, and at a 5% salal flower/berry solution (unbuffered) significantly reduced the germination value of Sitka spruce and western red cedar.  Watering seedlings with  the salal leachate solution resulted in total biomass 176  of Sitka spruce, western red cedar and western hemlock that was significantly lower than the control after 12 weeks.  The phenolic acid solution resulted in total  biomass of all seedlings to be lower than controls but only the Sitka spruce seedlings were significantly lower.  Longer term trials could produce more  significant results.  9.  The uptake of 32 P by mature roots was significantly reduced by the phenolic acid and salal leachate solutions  (unbuffered)  to 15% and 36% of controls  respectively for western red cedar and to 69% and 9% respectively for western hemlock.  10.  The uptake of 32 P by excised roots of seedlings which had been watered with the phenolic acid and salal leachate solutions  (unbuffered) were not significantly  different from controls for Sitka spruce and western hemlock.  This indicates that the phenolic effect is  reversible.  Uptake of inorganic P was actually higher  than the control,  indicating the seedlings were  probably deficient in P.  Uptake of inorganic P by  western red cedar was significantly lower than that for the controls,  indicating possible long term root  damage.  177  11.  Possible forest management techniques to improve site productivity on the CH phase may be to attempt to replicate the windthrow process using scarification to break up hardpans,  increase soil aeration, and improve  soil fertility by mixing mineral and soil horizons. The use of a nurse crop such as red alder, could improve the nitrogen balance of the site, add a large annual source of readily decomposable litter, and contribute to rapid shading of salal understorey. Fertilization with nitrogen and phosphorus is the best known silvicultural tool to relieve the growth—check of conifer regeneration.  2.0  CONCLUSION This study provides evidence to suggest that the growth-  check of conifer regeneration involves a number of interacting factors.  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