COLONIZATION of WOOD by MICRO-ORGANISMS a Thesis

COLONIZATION OF WOOD BY MICRO-ORGANISMS

A thesis submitted for the degree of Doctor of Philosophy of the University of London and for the Diploma of Membership of the Imperial College of Science and Technology

Colin Peter Clubbe B.Sc.

Department of Botany and Plant Technology Imperial College of Science and Technology London SW7 2BB

September 1980 2

FRONTISPIECE

TrichocZadium opacum sporing in soft-rotted birch fibre.

Successful or unsuccessful colonist?

Successful: Colonized and sporing inside the wood. Causing the soft rot? Therefore deriving nutritional benefit from the wood.

Unsuccessful: Dispersive spores produced in the centre of the wood with no access to the outside environment for effective dissemination.

ABSTRACT

The microbial ecology of small stakes of Pinus sylvestris and

Betula pendula, both untreated and treated with CCA, was investigated.

Two quantitative techniques were developed to monitor the

colonization and decay of wood in soil contact. The micro-organisms were

enumerated firstly by their isolation onto selective growth media and

secondly by direct microscopical analysis.

Fungi were identified as far as possible and assigned to

Ecological Groups. Six Ecological Groups were identified: Bacteria,

Primary Moulds, Stainers, Soft Rots, Basidiomycetes and Secondary Moulds.

The activities of the fungi in the wood were recorded using a microquadrat/veneer method. Sections were cut for light microscopy and small areas, or microquadrats, were scored for various colonization and decay characters. The data was analysed using a computer giving numerical expression to the direct observations.

Separately each technique only revealed part of the process.

Isolations gave no indication of the state of the organisms in the wood, whether spores or mycelium, nor their nutritional sources. From Direct

Observations identification of the fungi was virtually impossible. To- gether one substantiated the other and a more complete picture was obtained of the progression of the organisms and of their effect on the wood.

In untreated stakes Bacteria colonized first followed rapidly by

Primary Moulds and Stainers. The first decay fungi were Soft Rots which were quickly displaced by Basidiomycetes as the major wood-destroyers and climax to the succession. Secondary Mould colonization was associated with the initiation of decay.

In treated stakes the initial succession was similar. Soft Rots formed the climax, replacing the Basidiomycetes which did not colonize treated wood. Few Secondary Moulds were isolated. Soft rot attack occurred very rapidly in treated birch, but no decay was evident in treated pine. ACKNOWLEDGEMENTS

I would like to thank my supervisor, Dr. J.F. Levy, for his

enthusiasm and helpful discussions throughout my research and Dr. D.J.

Dickinson for his advice on preservation methods.

I am grateful to many of the technical staff of the Botany

Department for their assistance, but particularly to Mr. Ron Baker for

his tireless help in the preparation of the field stakes and for trans-

porting me to and from Silwood Park for my fieldwork.

I extend my thanks to all my colleagues in the Timber Technology

Section for their lively discussions and helpful criticisms of new ideas,

particularly to Mr. Ed Baines for his help in the development of the

computer programs.

For financial support I extend my gratitude to Rentokil Limited

and to Dr. P. Cornwall and Dr. C. Coggins for allowing me the use of

their facilities. Thanks are also due to Mr. K. Doughtey for his

assistance in treating the field stakes.

I am very grateful to Dr. B. Sutton and Dr. B. Brady of the

Commonwealth Mycological Institute for their help with fungal identifi-

cations and to Dr. A. Rayner for his comments on the basidiomycete cultures.

Two people have assisted directly in the production of this

thesis and I would like to thank them; Mr. Sinclair Stammers for his advice and assistance with the photography and Miss Suzanne Cheston for her speed and accuracy in typing the manuscript.

Finally I would like to thank Miss Deborah A. Seddon for proof- reading the manuscript and for her encouragement throughout my studies and during the preparation of this thesis. 6

CONTENTS

Page

TITLE 1

FRONTISPIECE 2

ABSTRACT 4

ACKNOWLEDGEMENTS 5

CONTENTS 6

SECTION 1: INTRODUCTION AND LITERATURE SURVEY 9 1.1 Aims and Objectives 10 1.2 Literature Review 14 1.2.1 Wood as a Natural Resource 14 1.2.2 Soil.Fungi: The Background to Decomposition Studies 15 1.2.3 Fungal Successions 17 1.2.3.1 The Concept of Succession 17 1.2.3.2 Successions of naturally occurring lignicolous resources 19 1.2.3.2.1 Living Trees 19 1.2.3.2.2 Dead Trees 20 1.2.3.3 Timber in Ground Contact 22 1.2.3.3.1 Untreated Timber 22 1.2.3.3.2 Preservative Treated Timber 25 1.2.4 Biochemistry and Micromorphology of Wood Decomposition 29 1.3 Design of the Field Trial 30 1.3.1 Timber Selection 30 1.3.2 Timber Preservation 31 1.3.3 Field Site 34 1.3.3.1 Choice of Field Site 34 1.3.3.2 Soil Data 37 1.3.4 Resin Coating 38 1.3.5 Physical Factors 41

SECTION 2: ISOLATION TECHNIQUES 42 2.1 Materials and Methods 43 2.1.1 Sampling Regime 43 7

2.1.2 Analysis of Stakes 43 2.1.2.1 Preparation of Samples 43 2.1.2.2 Isolation of Organisms 44 2.1.2.3 Selective Culture Media 48 2.1.3 Cultural Techniques 50 2.1.4 Sectioning of Inoculum Splints 51 2.2 Results 53 2.2.1 Description 53 2.2.1.1 2 Day Samples 53 2.2.1.2 4 Day Samples 54 2.2.1.3 8 Day Samples 54 2.2.1.4 16 Day Samples 55 2.2.1.5 32 Day Samples 57 2.2.1.6 96 Day Samples 59 2.2.1.7 184 Day Samples 62 2.2.1.8 265 Day Samples 65 2.2.1.9 363 Day Samples 67 2.2.1.10 500 and 591 Day Samples 70 2.2.2 Ecological Groups 72 2.2.3 Quantification 80 2,2.3.1 Untreated Birch 81 2.2.3.2 Treated Birch 84 2.2.3.3 Untreated Pine 84 2.2.3.4 Treated Pine 87 2.3 Discussion 90

SECTION 3: DIRECT OBSERVATION TECHNIQUES 101 3.1 Materials and Methods 102 3.1.1 Preparation of Material 102 3.1.2 Staining 104 3.1.3 Microscopy and Scoring Technique 106 3.1.4 Computer Analysis 121 3.2 Results 122 3.2.1 Pine 122 3.2.2 Birch 130 3.3 Discussion 142 3.3.1 Pine 143 3.3.2 Birch 153 3.4 General Discussion 159 8

SECTION 4: GENERAL DISCUSSION AND CONCLUSIONS 165

SECTION 5: IDENTIFICATION OF FUNGI FROM FIELD STAKES 175 5.1 Introduction 176 5.2 Materials and Methods 178 5.3 Results 185 5.3.1 Primary Moulds 185 5.3.2 Stainers 188 5.3.3 Soft Rots 191 5.3.4 Basidiomycetes 197 5.3.5 Secondary Moulds 200 5.4 Discussion 203

APPENDIX 1 Layout of Field Site 204

APPENDIX 2 Percentage Frequency of Isolation Tables 206

APPENDIX 3 Computer Program, Sample Data and Sample Output 214

REFERENCES 222 9

SECTION 1: INTRODUCTION & LITERATURE SURVEY 10

1.1 AIMS AND OBJECTIVES

The increasing need to conserve a diminishing resource requires information about the actual processes of colonization of wood by micro- organisms and how this could eventually lead to decay and ultimately to mechanical failure of the timber. The practical assessment of the decay of timber in ground contact has been based on operator experience and, at best, is a qualitative estimate of the service life of a given wooden stake, post or pole. The 'kick over' test for failure or the depth of penetration of a penknife have lead to '5 point' decay categories such as that used in the collaborative field trial of the IRG: 0 = Sound - no attack, I = Slight and superficial decay, 2 = Evident but moderate decay, 3 = Severe decay, 4 = Failure - almost complete loss of strength

(Dickinson, 1976). For many purposes such as the screening of large numbers of different species of wood, this type of assessment is perfectly adequate. In these instances a biannual or annual assessment of this type is easily applied and would distinguish between durable or semi- durable timbers of commercial interest and less important non-durable timbers, thus ranking timber species into categories of potential use.

Methods for preserving wood assumed greater importance and the established methods of decay assessment were applied to field trials containing a variety of wood species and different types of preservatives at a series of retentions. Again a ranking was important since any wood preservative which would not increase the service life of the timber by a significant length of time would be regarded as unimportant.

During the last twenty years attempts have been made to move away from these subjectively analysed tests and to evolve assessment methods which would give more information and early indications of the onset of decay. Ideally, the basis of these testing methods should Be quantitative, both in the collection of data and its analysis leading to

the possibility of predicting the course of events. With the build-up of 11 a sufficiently large pool of quantitative data predictive, mathematical modelling becomes a possibility, integrating details of wood species, type of preservative, microbial flora and decay patterns, with environmental factors such as soil type and moisture relations.

With these long term objectives in mind the immediate aim of the present work was to develop techniques by which wood-invading micro- organisms could be studied to elucidate their colonization patterns and the initiation of decay. A representative hardwood and softwood, both untreated and treated with a copper-chrome-arsenic (CCA) type preservative was used. The approach was bipartite, the first part involving the isolation and identification of colonizing organisms and the second observing the activities of these organisms at a cellular level using microscopic analysis of thin sections of wood.

The design of the main experiment was such that subjective assessment was eliminated as far as possible, thus allowing a more objective analysis based on sound statistical principles of the type used in higher plant ecology.

The problems of the analysis of wood-inhabiting fungi have been outlined with great clarity by Butcher (1971,a). Butcher pointed out the importance of the pattern of sampling, which could be subjective (selection of 'typical' zones) or objective (samples taken at random or systematically). To understand the decay process fully from initial colonization to final mechanical failure, it is essential to know when component members of the microflora first colonize the wood, what changes they undergo in quantity and distribution with time, any interspecific associations occurring within the wood and the effect of the micro-organisms on the wood structure. Butcher's analysis, using frequency as a measure of abundance, represented the first real attempt at objective analysis of objectively collected data. Earlier work in this field (inter aUa:

Corbett & Levy, 1963a, b; Merrill & French, 1966; Kaarik, 1967, 1968;

Banerjee 1970; Banerjee & Levy, 1971) have been described as subjective 12

analyses of objectively collected data (Dwyer & Levy, 1976).

The work of Dwyer and Levy was partially an extension of

Butcher's work, and involved the development of a veneer-quadrat sampling technique and a direct observation technique based on data collected from a model system of small, sealed stakes of wood buried in soil under controlled laboratory conditions. Dwyer and Levy (1976) summarized some preliminary results based on these laboratory studies and indicated the value of this combined approach to the study of wood decay.

The present work evolved from these preliminary studies of

Dwyer and Levy. The techniques were further developed and refined to observe colonization and decay sequences in the natural environment, namely that of wood in soil in the field and not in controlled laboratory conditions. The work was also extended to compare this natural ecological succession with a modified environment, that of preservative-treated wood.

In any ecological studies of this nature one must be aware of the limitations of the techniques employed. The important limiting factors have been summarized by Butcher (1971a) as:

1) The inability to examine a specimen more than once as sampling is necessarily destructive. 2) The inability to distinguish between active and dormant or moribund stages of the fungus. 3) The possibility that not all fungi can be isolated onto the particular medium (or media) used at the particular conditions of incubation. 4)

The lack of any visual check that may be made on the accuracy of the sampling. To these could be added: 5) The lack of details on the functional significance of the isolates in situ.

Points two tō five relate directly to the difficulty of finding any technique that will enable an accurate assessment of the course of events between wood being placed in the soil and its destruction by decay.

The main problems of isolation work are maintenance of sterility, 13

contamination, optimum nutrient, temperature and oxygen for growth of the organisms and their reproductive bodies and how to measure them.

With these limitations in mind the Direct Observation and Isolation

Techniques were developed within an ecological framework. 14

1.2 LITERATURE REVIEW

1.2.1 WOOD AS A NATURAL RESOURCE

In the natural environment wood is an identifiable component

of the detritus entering the soil ecosystem and as such forms a resource

for the decomposer organisms inhabiting the soil. Swift et al. have

suggested the use of the term 'resource' in preference to the more widely

used 'substrate' since it infers none of the biochemical specificity

associated with enzyme-substrate complexes etc. and can refer to any

organic material entering the decomposer subsystem (Swift, 1976, 1977;

Swift, Heal & Anderson, 1979). Decomposition essentially results in a

change of state of the resource under the influence of a number of biotic

and abiotic factors. This generally results in a decrease in mass of

that resource. Catabolism results in organic intermediates derived from

the wood entering the decomposer organisms and being resynthesised into

decomposer tissue. Some products will be inorganic and recycled princi-

pally as part of the nitrogen and phosphorus cycles. Organic products

eventually become part of the carbon cycle.

It is these natural decomposer cycles which need to be interrupted in order to use wood as a structural material. Untreated wood forms a primary resource for the decomposer organisms. Wood treated with a preservative forms a modified environment which may change the patterns of colonization and decay by selecting resistant organisms which may eventually be able to fully colonize and decompose the wood in spite of the chemicals added.

It is these considerations which must be appreciated when developing techniques for the analysis of wood and the micro-organisms which inhabit it, to understand fully their interactions and how and when decay occurs before steps can be taken to delay it sufficiently or completely stop the decay process. 15

1.2.2 SOIL FUNGI: THE BACKGROUND TO DECOMPOSITION STUDIES

Almost one hundred years ago Adametz (1886) initiated the study of soil-inhabiting fungi when he described the microflora of sandy and loamy fields by inoculating sterilized media with soil from the surface and from some depth. From these he obtained eleven species of fungi and these represented the first fungi isolated from the soil. Oudemans and

Koning (1902) greatly improved the isolation techniques identifying some forty species of fungi. However, the main interest was still centred around the bacteria, particularly in association with the nitrogen cycle

(Beijerinck and Winogradsky, 1891-1910). As late as 1902 when Omeliansky published his studies on the anaerobic cellulose bacteria, no consideration was given to the role of fungi or other micro-organisms in this process. Possibly prompted by this lack of information Waksman posed the question, "Do fungi actually live in the soil and produce mycelium?"

(Waksman, 1916a). His initial work in this area (Waksman, 1916b, 1917,

1922a, b, 1927) encouraged many other studies aimed at elucidating the nature of the fungal flora of the soil. Most of this early work was concerned purely with floristic data and little attention was paid to the ecological aspects of fungal growth in the soil. As a result of extensive surveys of soil fungi these early workers concluded that the soil contained a constant and characteristic fungal flora.

During the last sixty years a great array of techniques have been evolved to evaluate the soil mycoflora both qualitatively and quantitatively (see Warcup, 1960, 1967; Parkinson, Gray & Williams 1971; Barron,

1971; Johnson & Curl, 1972). Initially most of the investigations dealing with soil-inhabiting fungi were based on essentially bacteriological methods involving dilution of soil suspensions. However, many workers began developing techniques specifically for the isolation of fungi from soil, recognising it as a separate problem requiring new methodology and not just the modification of existing ideas. Many of these techniques are 16

routine methods today, such as the Rossi-Cholodny slide (Rossi, 1928;

Cholodny, 1930), the immersion-tube method (Chesters, 1940) and Warcup's hyphal isolation method (Warcup, 1955). Together with the development of more selective methods, particularly 'baiting' techniques and selective growth media, much information has accumulated on the fungal flora of the soil. A number of these techniques have been modified and applied to the more ecological activities of the mycoflora, particularly in association with litter breakdown, both on the surface and within the soil. However, any method used to study the presence and activities of fungi in a particular habitat will influence the results. Much of the difficulty has centred around the fact that fungi may exist in soil either as mycelium or spores. Isolation techniques are selective, to a greater or lesser extent, and cannot distinguish between active mycelium and resting bodies whereas direct observation techniques give little information as to the identity of the observed organism (Garrett, 1955). Harley and

Waid (1955) re-stated the problem, saying that one of the first requirements for any ecological study of soil organisms is that the methods used must distinguish between organisms which are vegetatively active and playing a part in soil processes and those which exist in a dormant or inactive form as spores or other propagules. These difficulties have been discussed most informatively by Warcup (1965). These comments are equally valid for fungi inhabiting soil inclusions such as wood and leaves, as they are for the soil itself.

The picture of the soil that has emerged is of a three-dimensional system of organic microhabitats dispersed through an essentially mineral matrix. Each microhabitat has its own microflora which may be active or dormant (Pugh, 1974). This system may be interrupted by living material such as roots on which a very specialised flora develops, the rhizosphere, or dead material in the form of litter. In order to exploit the dead substrates fully the fungi must be good competitive saprophytes (Garrett, 17

1951). The ability of a fungus to grow on a substrate depends upon its possession of the necessary enzyme systems, favourable environmental conditions and its competitive ability, a balance of these factors determining its role in decomposition. During decay the substrate changes continuously and this, together with interactions between organisms results in progressive changes in the micro-organisms involved in the decay process.

1.2.3 FUNGAL SUCCESSIONS

1.2.3.1 THE CONCEPT OF SUCCESSION

Plant ecology involves the study of vegetation and endeavours to unravel the complex inter-relationships between individuals and groups of plants which form the community. In the same way microbial ecology, as a branch of ecology, is concerned with the inter-relationships of micro-organisms occupying a common habitat or series of habitats as part of the soil. The phenomenon of autogenic succession, by which a series of transitional plant associations colonize an area, altering the conditions such that other, more demanding, species displace them until a stable climax is reached is well known in higher plant ecology. Success- ion can similarly be seen in microcosm in the colonization and utilization of a substrate by soil micro-organisms (Garrett, 1955). Whereas a large number of plant successions have been described (see Kershaw, 1974), the study of fungal communities lagged behind. This led Garrett to undertake a comprehensive review of substrate relationships of fungi, particularly the economically important root-infecting fungi (Garrett, 1950, 1951,

1956). Garrett (1951) proposed the study of soil fungi as ecological groups as a convenient approach to the investigation of the relationships between fungi and their habitats. If a root died through natural sen- escence Garrett suggested that the pioneer colonists were likely to be weak parasites or obligate saprophytes of the 'sugar fungi' group.

Earlier Burges (1939) had proposed the existence of a specialised group 18

of 'sugar fungi' which were capable of Utilizing readily available sugars and pentosans, but not cellulose or lignin. These 'sugar fungi' then opened the way for cellulose decomposers and finally lignin decomposers to invade the tissue. These observations on root-infecting fungi prompted first Garrett (1963) and latterly Hudson (1968) to attempt a generalised scheme of fungal succession on plant tissue lying within or upon the soil.

During this succession the substrate becomes progressively depleted, first of sugars and simpler carbon compounds, then cellulose and finally lignin.

Garrett also recognised a secondary group of sugar fungi which did not live on the simple sugars initially present in the wood, but on the breakdown products of cellulose-decomposing fungi. These 'secondary sugar fungi' thus appeared in association with the decay fungi.

This generalized scheme for fungal succession on litter can be summarized as follows (Figure 1).

Living Tissue Senescent Tissue Dead Tissue Stage la Stage 1 Stage 2 Stage 3

Weak parasites Primary sapro- Cellulose de- Lignin decom- (Ascomycetes phytic sugar composers and posers (Basidio- and Fungi Im- fungi. (Asco- associated mycetes) and perfecti) mycetes and secondary sa- associated fungi May be host Fungi Imper- prophytic (Secondary specific. fecti) Living sugar fungi Moulds) on sugars and utilizing carbon com- products of pounds simpler cellulose de- than cellulose. composition (Ascomycetes and Fungi Im- perfecti, possibly Bas- idiomycetes)

Figure 1: Generalized scheme for fungal succession on litter (after

Garrett and Hudson) 19

Many successions investigated agree generally with this scheme,

but several deviate from it.

In a survey of interactions between fungi D'Aeth (1939) ex-

pressed the view that successional diseases occurred on the living host

through the action of one fungus developing a substrate which was more fāvourable for a second fungus. Thus in a pioneer community the most aggressive parasite would invade and alter the substrate allowing the

entry of other organisms which interacted before the most virulent one continued the invasion.

1.2.3.2 SUCCESSIONS ON NATURALLY OCCURRING LIGNICOLOUS RESOURCES

1.2.3.2.1 LIVING TREES

One of the most comprehensive accounts of the successions of fungi colonizing living trees is that of Etheridge (1961) on branch infections of PopuZus tremuloides. The succession was one of bacteria - non-decay fungi - primary and secondary decay fungi, with the first

Basidiomycetes appearing only after 8-9 years. Essentially similar successions were found on Populus trunks by Good and Nelson (1962) and Shigo

(1963). Successions on conifers have exhibited the same pattern as those on hardwood (inter alfa: Bourchier, 1961 on Pinus contorta and Whitney,

1962 on Picea giauca). Basidiomycetes are usually late colonizers, but it has been shown that under certain conditions they can be pioneer colonizers, attacking forest wood before other organisms. Pomerleau and

Etheridge (1961) found Stereum sanguinolentwn to be the pioneer colonist on branch wood of Abies balsamea. Heterobasidion (Fomes) annosus can be the first colonist on pine stumps (Rishbeth, 1950, 1951). Polyporus tomentosus and Stereaan spp. have also been regarded as pioneer invaders

(Etheridge, 1961; Whitney, 1962). However, in these cases the basidiomycete fungi tend to be acting as weak parasites and would thus fall into

Stage la of Garrett's scheme, then initiating decay immediately without the successional development. 20

Kāārik (1974) summarized some general trends of saprophytes colonizing living trees commenting that the invasion of wood by decay fungi following wounding is usually preceded by bacterial attack and colonization by non-decay fungi. The decay fungi are thus secondary colonizers of previously occupied tissue. This succession seems to be common to both hardwoods and softwoods, although individual species may be different. However, some decay fungi can be pioneer colonizers of wounded tissue, but they tend to decline if the wood is colonized by other organisms. Kaārik (1974) also reviewed the other aspects of colonization of living trees, viz. successions following insect attack, in fire-scars and following parasitic attack. With the exception of that following parasitic attack, which is not clearly established, successional trends in the others were similar to those already described. The subject of decay in standing trees has more recently been reviewed by

Mercer (1980).

Decay in living trees is characterized by a response of the tree to the invading organisms. The first response is probably electrical, then chemical to limit the area of infection (Mercer, 1980). This forms a 'reaction zone' between healthy and discoloured wood. Shigo (1976) has proposed the term 'compartmentalisation' to describe this cellular reaction.

1.2.3.2.2 DEAD TREES

Once the tree is felled it rapidly dies due principally to dessication. No response to invasion is therefore possible and decay is an ecological consequence and not a pathological problem.

Two pioneer studies on the successions of organisms on dead wood were conducted by Chesters (1950) and Migenot (1952). In his pre- sidential address to the Lincolnshire Naturalist Union, Chesters (1950) outlined the succession of microfungi associated with the decay of logs and branches of deciduous trees. The primary colonists were wound 21

parasites which were responsible for the shedding of the branches, or aggressive saprophytes which attacked moribund branches. These were mainly host-specific Ascomycetes such as Hypoxylon and Diatrype. Basi- diomycetes such as Coriolus (Polyporus) versicolor and Stereum hirsutunt were also found as primary colonists and these may be thought to be

equivalent to Garrett's stage la. Once the primary phase of colonization was well advanced succession depended upon internal and external factors, of which moisture content of the wood and the nature of the remaining

nutrients were the most important.

A detailed ecological study of the succession of fungi on

deciduous woods was carried out by Mā enot (1952). Groups of fungal

colonists were found to belong to a particular 'stage' in decay, each.

stage being named after the most common fungus in the group. Although

seven stages of decomposition were distinguished by Maenot, they have

their analogues in Garrett's general scheme. Thus the Phellinus stage

(1),with Basidiomycetes and Ascomycetes attacking the living tree and

remaining active after death of the tree for a variable period of time,

is parasitic (equivalent to Garrett's stage la). The Phialophora

fastigiata stage (2) is equivalent to Garrett's stage 1 of primary colon-

ization of dead wood. The Melanoma (3) and Mortierella ramanniana (4)

stages characterize the initiation of decomposition of the wood itself

(cellulose breakdown of Garrett's stage 2). The 5th stage in Mgenotts-

sequence, the Leptoporus stage, included species of Basidiomycetes capable

of lignin breakdown (equivalent to Garrett's stage 3). The final two

stages of Magenot's scheme, the Mollisia stage (6) and the Bisporomyces

stage (7), are the advanced stages of decomposition (stage 6) including

chitin-decomposing fungi; and those parasitic or saprophytic on other

fungi (stage 7) .

Basham's comprehensive account of the fungal successions involved

in the decomposition of fire-killed pines in Canada (Basham, 1957) showed 22

that staining fungi were the first to attack the sapwood before it was invaded by decay fungi, particularly PhZebia (Peniophora) gigantea and

Hirschioporus (Polyporus) abientinus. That staining fungi preceded decay fungi was also shown by Ueyama (1966) on logs of Fagus crenata in Japan.

On unpeeled logs of birch and aspen intended for pulpwood,

Henningsson (1967a, b, c) found that organisms with low wood-destroying activity such as Trichoderma and some bacteria were early invaders and wood-destroying Basidiomycetes appeared much later. Similar studies of the colonization of pulpwood bolts have yielded comparable results

(Findlay, 1940; Atwell, 1956; Shigo, 1962).

1.2.3.3 TIMBER IN GROUND CONTACT

1.2.3.3.1 UNTREATED TIMBER

With this background of ideas and techniques derived from fungal successional studies in the natural environment several workers began to look at the biodeterioration of wooden, debarked posts in contact with the soil. The early work in this area (inter aZia: Corbett & Levy, 1963a, b; Merrill & French, 1966; Kadrik, 1967, 1968; Levy, 1968; Butcher, 1968a, b; Kerner-Gang, 1970; Banerjee, 1970; Banerjee & Levy, 1971) was princi-

pally concerned with detailing the organisms involved in the colonization and decay sequences, their position, and role within that sequence.

Corbett and Levy (1963a, b) suggested a pattern of colonization of Monil.iales Gp. I (including Trichoderma viride, Penicillium spp., and

Botrytis sp.) - Sphaeropsidales (including several soft rot fungi) -

Moniliales Gp. II (including Cylindrocarpon sp.) - Basidiomycetes. This

sequence was based on isolations from fence posts of Betula sp. and

Pinus sylvestris. In the United States a comparable study on Pinus

ponderosa stakes in soil contact by Merrill and French (1966) showed a

similar pattern of colonization. The early colonists were mainly of the mould-type with Fusarium solanii, Trichoderma viride, Aspergillus ustus

and Penicillium spp. accounting for 92% of all isolates. Soft rots 23

became established between six and twelve weeks and finally a Basidio- mycete, Sistotrema (Trechiospora) brinlonanii was isolated after twelve weeks. Sistotrema brinlonanii was found to be the most important organism degrading lignified material in the soil in France by Magenot and

Reymond (1963). However, the ability of Sistotrema brinknanii to attack lignin and its general role in colonization have recently been questioned as a result of isolations from external joinery (Baker et al., 1979;

Carey, 1980).

The succession of fungi attacking spruce and pine poles in three different forest soils. in Sweden was studied by Kāārik (1967, 1968).

Isolations were made after six, twelve and eighteen months. At six months the poles showed locally restricted attack by different Basidiomycetes,

Peniophora pithya and Stereum sanguinolentum being particularly abundant.

In addition many Fungi Imperfecti and Ascomycetes were isolated. These included moulds such as Trichoderma viride and sap-stains such as

Ceratocystis pilifera. As the earliest isolations were carried out after six months it was impossible to establish an order of colonization. After a further six winter months no obvious changes occurred, but after a further six summer months attack by Basidiomycetes was heavy with Phiebia

(Peniophora) gigantea, Climacocystis (Polyporus) borealis and Stereum sanguinolentum occurring in particularly high frequencies. Changes also occurred in the non-basidiomycete flora with many of these probably acting as Secondary Moulds (Clubbe,1978).

Butcher (1968a) described the succession of fungi colonizing untreated stakes of Pinus radiata sapwood. At the groundline the succession of organisms was complete: primary moulds - soft rot fungi - secondary moulds and Basidiomycetes. Butcher also looked at the above and below-ground zones and showed that succession proceeded more slowly below ground than at the groundline principally because of the higher moisture content and also because colonization was restricted to soil-borne fungi. 24

The low moisture content above ground limited colonization to air-borne blue-stain fungi and moulds and did not get to the stage where decay fungi became established.

Banerjee and Levy (1971) described the colonization of fence posts of Betula sp.and Pinus sylvestris. They showed the importance of bacteria as primary colonists of groundline tissue. The surface layers of the birch were then colonized by rapidly-growing mould fungi, Fusarium spp., Botrytis sp. and Mucor sp. being very common. After a time bluestain fungi such as Ceratocystis piceae and soft rots such as Phialophora spp. and Chaetomium dolichotrichum were'isolated below the surface and later at some depth. Basidiomycetes, including Polystictus sp., were first isolated after four months, appearing at depth, but not at the surface. The sequence in pine was essentially similar with some slight differences in species composition.

The work on timber in ground contact inevitably led to the development of techniques specifically designed for isolating fungi from wood, with all its inherent difficulties. Greaves and Savory (1965) discussed the use of four methods of isolation: the Pressler increment borer, the two-chisel technique, the sterile-block technique and the saw-cut method. A drill technique was developed by Levy (1967) and a modification of this used in his fence post studies (Levy, 1968). Other techniques developed include the split-block technique (Kdārik, 1967,

1968), the grinding-wheel technique (Eggins, Malik & Sharp, 1968) and the split-billet technique (Shigo, 1965). Many of these methods have been reviewed by Grant and Savory (1968). These techniques were primarily developed with the object of obtaining pure cultures of individual species and the maximum number of different species. For objective analysis, however, it is desirable to employ techniques where a standard volume of wood inoculum is used and none of these techniques satisfy that condition.

Butcher (1971a) has also commented on the effects of different sized inocula. 25

The sampling techniques of Dwyer and Levy (1976) were the first to achieve a standard inoculum volume allowing rigid statistical analysis.

1.2.3.3.2 PRESERVATIVE TREATED TIMBER

As the techniques for the isolation of fungi from wood were refined the scope of the work expanded to include preservative-treated wood which had received little attention prior to Butcher's systematic study of Pinus radiata stakes treated with a CCA preservative to low retention (Butcher 1968b, 1971b). Using the two-chisel technique (Greaves

& Savory, 1965) that he had used for untreated stakes (Butcher, 1968a)

Butcher followed the succession of fungal species colonizing the stakes over a two-year period in above-ground, ground-line and below-ground zones. The succession at the ground line was moulds - soft rots - secondary moulds and primary Basidiomycetes (white rot fungi), the same as he had encountered in untreated material. Basidiomycetes became

established after the stakes had been in the ground for 13 months and although decay was evident below groundline, it was never observed at the groundline. The isolated Basidiomycetes were tested for copper tolerance

and, with the exception of Poria vaz1lantii, were found to be inhibited by

very low levels of copper. The Basidiomycetes were much more sensitive

to copper than were the early colonists. It would seem that the very low -3 levels of CCA used (1.52 - 2.4 kg m ) had little effect on the success-

ion of fungi, although the appearance of the Basidiomycetes was delayed

by nine months. The levels of copper in the wood by this time must have

been below the toxic threshold of the Basidiomycetes.

In order to be a successful primary colonist of treated wood the

fungus must possess a tolerance to the preservative used. Although infor-

mation is becoming available as to the tolerances of particular species of

fungi little is known about the moulds and stainers as a whole. There is

evidence that many of the mould and stain fungi can tolerate relatively

high concentrations of multisalt preservatives (Duncan 1960). Duncan and 26

Deverall (1964) have reviewed the early work on detoxification of preservatives showing that some fungi were capable of degrading the toxicant into a less potent form, thus rendering it less effective in protecting the wood from decay by the less-tolerant basidiomycetous wood-destroyers.

Microfungi, particularly soft rot fungi, are generally more tolerant to wood preservatives than are Basidiomycetes (Savory, 1955; Duncan, 1960;

Da Costa & Kerruish, 1963; Butcher,1971b; Henningsson & Nilsson, 1976a).

In general basidiomycete fungi are very sensitive to wood preservatives although certain species or strains of Basidiomycetes do show some re- sistance (Zabel, 1954; Baechler & Roth, 1956; Cowling, 1957; Gersonde,

1958; Young, 1961; Da Costa & Kerruish, 1964; Butcher, 1971b). Findlay and Savory (1950, 1954) and Savory (1954, 1955) had earlier indicated that copper was the main toxicant in the water soluble copper-chrome- arsenates and this has more recently been confirmed by Hulme and Butcher

(1977b).

Stranks and Hulme (1976) discussed possible mechanisms for the biodegradation of wood preservatives by microfungi. This detoxification involved valence changes and alkylation of inorganic wood preservatives such as the CCAs ; hydroxylation, ring cleavage and oxidation for aromatics such as pentachlorophenol; and terminal oxidation and s-oxidation for creosote-like preservatives. Work on the detoxification of some preservatives used in external joinery is currently being conducted at the

Princes Risborough Laboratory (Henshaw et al., 1978; Carey, 1980).

The importance of mould and stain fungi as early colonists of

CCA - treated wood has been shown by Greaves (1972). Greaves also stressed the possible importance of bacteria and Actinomycetes as early colonists. Soft rot fungi became established after four months in both untreated Pinus radiata and Eucalyptus regnans and also in the Eucalypt stakes treated to 4.0 kg.m 3. By the end of seven months soft rot fungi were present in all but the highly treated pine stakes (12.0 kg.m 3). 27

Although Basidiomycetes were not isolated from any treated stakes they were occasionally seen in sections as hyphae with clamp connections.

Soft rot attack is now recognized as the most important problem in preservative-treated wood since in most situations preservation gives good protection against basidiomycete attack. The problem is world-wide, but .notably in tropical climates. For example, in Queensland, Australia it is estimated that up to 400,000 CCA-treated Eucalyptus transmission poles are affected to varying degrees by soft rot (Greaves, 1977). In

Sweden up to 4 million 20-35 year-old salt-impregnated transmission poles of Pinus sylvestris are attacked by soft rot (Friis-Hansen, 1976). There are few reports of Basidiomycetes in preservative-treated wood (Henningsson

& Nilsson, 1976a).

The term 'soft rot' was proposed by Savory (1954) for decay caused by cellulose-destroying microfungi to distinguish them from the brown and white rots caused by the wood-destroying Basidiomycetes. How- ever, this was almost one hundred years after the German botanist Schacht

(1850, 1863) observed and illustrated soft rot cavities in plant cells.

Isolations from preservative-treated wood have been made by several authors and the isolations have yielded a number of soft rot organisms (Findlay & Savory, 1954; Savory, 1954; Duncan, 1960; Courtois,

1963; Greaves & Savory, 1965; Kerner-Gang, 1966; Gersonde & Kerner-Gang,

1968; Butcher, 1971b; Greaves, 1972). Henningsson and Nilsson (1976a, b) noted that the microflora of treated telegraph poles in Sweden exposed for 20-35 years was dominated by microfungi, with few Basidiomycetes isolated. Soft rot attack was evident in the poles and most of the isolated microfungi showed cellulolytic activity in culture and were able to attack the wood through cavity formation or cell wall erosion. Many showed a high tolerance to copper, zinc and arsenic.

Fougerousse (1976) investigated the spectrum of fungi involved in the soft-rot attack of treated stakes in field tests in the Ivory Coast 28

and France, indicating the importance of the genera Phialophora and

Rhinocladiella. The importance of Phialophora species was further highlighted by Nilsson and Henningsson (1977) who compiled the known occurrences of Phialophora species occurring in preservative treated wood in ground contact as reported in the literature. The Phialophoras have also been shown to be the dominant floral components of CCA-treated Eucalyptus power transmission poles in Australia (Leightley, 1978, 1980), and of

CCA-treated test stakes in England (Clubbe, 1978, 1980).

The soft rot fungi, particularly Phialophora species, are of greatest importance in preservative-treated timber, a situation in which the principal wood-destroying fungi, the Basidiomycetes, are excluded.

Many microfungi are capable of causing soft rot. Seehann, Liese and Kess

(1975) have listed 305 true soft rot fungi, representing 172 genera, capable of producing cavities or erosion in woody tissue.

Much work is now underway in research laboratories across the world into the 'soft rot problem'. The reasons for the basic differences in susceptibility between treated hardwoods, which generally perform badly, and treated softwoods, which perform well, are being investigated

(Butcher, 1980; Greaves, 1980). The variability in the performance between different hardwood species is also under investigation (Aston &

Watson, 1976; Hulme & Butcher, 1977a; Liese & Peters, 1977; Butcher, 1978;

C. Levy, 1978). Much of the work involves examination of the microdistribution of the preservative elements across the wood cell walls

(Dickinson & Sorkhoh, 1976; Dickinson, Sorkhoh & Levy, 1976; Greaves &

Levy, 1978; Levy & Greaves, 1978; Drysdale, 1979).

The search for a new preservative is a very lengthy process and any rapid screening technique which can give a quick indication of the potential of a new chemical or formulation will be of enormous benefit.

Thus, many laboratories are developing bioassay techniques for screening potential wood preservatives as shown by the number of contributions to 29

the recent special seminar organized by the International Research Group on Wood Preservation at its 10th Annual Meeting (IRG, 1978).

1.2.4 BIOCHEMISTRY AND MICROMORPHOLOGY OF WOOD DECOMPOSITION

Thus, decay of wood results from the destructive activity of certain fungi, principally white, brown and soft rots. The action of extracellular enzymes secreted by these fungi results in primary catabolism of the various components of the wood cell wall (see Green,

Dickinson & Levy, 1980; Kirk, 1980; Montgomery, 1980). These breakdown products enter the metabolic pool of the decomposer organisms where they are re—synthesized into the polysaccharides and proteins of the fungal tissues. This allows growth of the vegetative hyphae which ramify through the wood, penetrating and eventually destroying the cell walls.

The mechanisms of attack and the micromorphology of the decay patterns are complex and have been the subject of much work (inter alio: Proctor,

1941; Wilcox, 1968; 1970; Levy, 1965, 1980; Liese, 1970; Zainal, 1975).

This whole area has recently been reviewed by Crossley (1979) in relation to his electron microscope studies on the three major types of decay and the reader is referred to that for any further details. 30

1.3 DESIGN OF THE FIELD TRIAL

1.3.1 TIMBER SELECTION

A representative British hardwood, Betula pendula Roth (silver birch), and a native softwood, Pinus sylvestris L (Scots pine) were used in the present study. These species were chosen because both have been the subject of study at Imperial College and elsewhere for some time and thus a large amount of background information was available to help in- terpret the results. Dickinson and Sorkhoh (1976) had previously used birch and pine in laboratory studies and found differences in behaviour, particularly in the CCA-treated blocks. To some extent these two wood species were used to see if these differences would be repeated under field conditions. Freshly-felled logs were flat sawn into planks and kiln dried before selection took place. Only the sapwood of both species • was used. This ensured a more uniform test and a more severe one, since sapwood is generally less durable than heartwood. This also relates to normal practice where a greater proportion of sapwood is now used by the timber industry.

Distinction between heartwood and sapwood of Pinus sylvestris was achieved by the use of o-anisidine (2-methoxyaniline) as described by Stalker (1971). Direct brush-application of the reagents was found more satisfactory than spraying as suggested by Stalker. No such straightforward test exists for birch and there is no colour difference between heartwood and sapwood. Many chemicals were tested to see if any differences could be detected and although a measure of success was achieved using iodine in potassium iodide (I2KI), possibly due to residual starch in the axial and ray parenchyma cells of the sapwood (see Plates 4.5 &

4.6) no reliable, reproducible technique was found. Thus to ensure that only the sapwood of birch was used, stakes were cut from wood not more than 60mm. below the bark.

The sapwood was cut into small stakes whose final size was 31

30 x 45 x 250mm. This optimum size was decided upon from both practical considerations and the use to which samples were to be put. All the stakes were orientated such that the 30mm. faces represented true tangential longitudinal faces and the two 45mm. faces were as close as possible to radial longitudinal faces (Plate 3.1).

1.3.2 TIMBER PRESERVATION

Chemical wood preservatives even when chosen and applied correctly to timber in ground contact are not necessarily able to eliminate micro- organisms from the treated wood. They may simply delay the onset of colonization and decay or they may select resistant strains or species from the microflora which are normally out-competed, except in this specialized environment.

In order to investigate the effects of the introduction into the wood of potentially toxic chemicals on the pathways of colonization and the microflora associated with this new habitat, half the timber stakes in the study were treated with a water-borne preservative of the copper- chrome-arsenic (CCA) type. Thus the performance of treated timber could be assessed along side that of untreated timber.

The stakes were vacuum-pressure impregnated with a 1% solution of Celcure A using a Bethell Process to give a net dry salt retention of

8.17 ± 0.29 Kg.m 3 in pine and 7.47 ± 0.40 Kg.m 3 in birch. Celcure A is a type 2 CCA (as described in BS 4072, 1974) and the formulation used was:

CuSO4.5H20 : 30%

Na2Cr207 : 33.4%

H3As04 (80%) : 32%

Water : 4.6%

The treatment was carried out in the pilot plant of the Rentokil Research

Laboratories, East Grinstead, (Plate 1.1.). 32

PLATE 1

Treatment of stakes with CCA

1.1 Pilot plant at the Rentokil Research Laboratories.

Stakes were vacuum, pressure impregnated using a Bethell Process.

1.2 Wet-fixation of CCA-treated stakes.

The air in the polythene bag was replaced by xylene vapour (from

xylene in beaker) to prevent the growth of slime moulds.

1.3 Dry-fixation of CCA-treated stakes.

Spacer-sticks between rows provided adequate ventilation around

stakes.

After treatment the preservative was fixed in the cell wall by

a fourteen day wet fixation during which time the stakes were left in

contact with the preservative fluid in a sealed environment (polythene

bag), the air being replaced by xylene vapour to prevent the growth of

slime moulds (Plate 1.2). This was followed by a further fourteen days

dry fixation in the open air (Plate 1.3). The stakes were then fully

leached using the 'water exchange method' (Savory, 1972). This involved

maintaining the stakes in five times their own volume of distilled water

for a period of fourteen days. The water was changed after 24 hours and

48 hours and then a further seven times with a minimum of 24 hours and a

maximum of 72 hours between each change. Finally, all the stakes were

air dried before being buried, their moisture content then being about

12%.

1.3.3 FIELD SITE

1.3.3.1 CHOICE OF SITE

A site was required of suitable size to accommodate 500 stakes.

Ideally the site should be as uniform as possible, well drained, agricultural land which had been undisturbed for a sufficiently long period to enable a reasonably wide, natural microflora to develop into a stable community.

A suitable site, subsequently called the Lodge Site, was selected at the Imperial College Field Station, Silwood Park near Ascot. This was large enough to accommodate the present trials with sufficient extra area available for a four—fold expansion, if necessary. It is well drained, with a slight run—off from west to east, of good fertility and it had been left fallow for ten years. The site is exposed, being on a slight hill, although some cover along the western edge is provided by a line of oak and birch trees (Plates 2.1 & 2.2). 35

PLATE 2

The Lodge Site at Silwood Park

2.1 The undisturbed site before the beginning of the field experiment

(October 1976).

2.2 The site with untreated and treated stakes in position consisting

of 20 rows of 25 stakes. (After the first sampling, October 1976).

1.3.3.2 SOIL DATA

Soil Type: Sandy loam

Humus Content: 6.06 ± 0.14% Water Holding Capacity: 21 ± 0.5%

p.H. (with d.H20): 6.38 ± 0.06

(with 0.05M CaC12):5.72 ± 0.01

Elemental composition of total soil using radiofrequency argon plasma spectroscopy.

Concentration Standard Element (ppm) Deviation

Aluminium (Al) 8233 621 Barium (Ba) 33.00 1.22 Boron (B) 20.56 11.94 Cadmium (Cd) Not Detected - Calcium (Ca) 2668 203 Chromium (Cr) 53.73 8.14 Cobalt (Co) Not Detected - Copper (Cu) 16.30 1.28 Iron (Fe) 11217 763 Lead (Pb) 48.00 15.12 Magnesium (Mg) 747 57.35 Manganese (Mn) 161.25 11.01 Nickel (Ni) 7.67 1.04 Phosphorus(P) 365 35.94 Strontium (Sr) 28.38 1.57 Vanadium (V) 31.23 2.13 Zinc (Zn) 50.50 3.07

(Luton, 1978 Pers. Comm.)

Cation exchange capacity: 13.46 ± 1.36 milli equivalents Na /100g soil 38

1.3.4 RESIN COATING

Immediately prior to burial all stakes, both treated and un-

treated, were coated on three longitudinal sides with an epoxy resin

sealant of the Araldite type, leaving the outer tangential face and two

ends unsealed. The sealant used was a combination of two resins and two

hardeners, all supplied by Ciba-Geigy (UK) Ltd. Plastics Division,

Cambridge. These were mixed in the following ratio:-

Resins: GY250 : 41 parts per hundred (pph) GY298 : 27 pph

Hardeners: HY830 : 8 pph HY850 : 24 pph

These ratios were calculated from the technical data in order to achieve

the best combination of hardness and durability with flexibility (Ciba-

Geigy Technical Bulletin TB 4.4).

The outer tangential face was left unsealed, open to the environ-

ment (Plates 3.1 & 3.2). This gave a means of access to micro-organisms

and enabled the progressive colonization in depth which occurred radially

to be monitored with time. It also afforded a model system for the full round pole in service which, of course, has only the outer tangential face exposed.

Each stake was given a number and with the use of random number

tables (Fisher & Yates, 1957) individual stakes were assigned to a particular position within the field site (see Appendix 1). Because of this randomization there were no constraints as to where the different treatments were to go within the area in question. This experimental design assured an unbiased estimate of sampling variance, thus ensuring more valid comparisons between treatments. To ensure a greater validity of between treatment comparisons all stakes were sampled as matched pairs

(Plate 3.3). Thus, during the original preparation of the wood, stakes were cut 500mm. long. These were then cut in half, one of which was 39

PLATE 3

3.1 A single, untreated Scots pine stake in situ. The orientation and

resin coating are clearly visible.

3.2 A single, untreated birch stake in situ, showing the maximum

extent of coverage by vegetation. The grass was cut when it

reached this stage.

3.3 Sampling of matched pairs.

An untreated (U60) and treated (60) Scots pine stake just after

removal from the soil showing the groundline marked in the resin (g).

treated and one left untreated. These were given the same number and

sampled as matched pairs (60 and U60 in Plate 3.3).

Each stake was buried to a standard depth of 150mm. (Plates 3.1

& 3.2) .

1.3.5 PHYSICAL FACTORS

The availability of water is the single most important factor governing the entry and colonization of micro-organisms into the wood.

For colonization to take place once the stakes were placed in the ground their moisture content must be increased to fibre saturation point (approximately 30% moisture content). The wood, being half-buried acts as a wick, having a permanent evaporative surface above the ground (Baines & Levy,

1979). The stake thus exerts a force upon the soil water. The force with which the soil retains its water is known as the water potential (pF) which is a measure of the availability of water. If the soil is wet its own affinity for water is low and thus water moves from the soil to the wood. Thus, by knowing the pF of the soil the amount of water available to the timber and consequently to micro-organisms can be inferred.

The initial pF of the soil was measured prior to stake burial and as each stake was sampled the pF of the soil adjacent to the exposed tangential face was also measured. This involved taking soil samples back to the laboratory for analysis. The Filter-Paper Method for determining the pF of the soil was used throughout as described by Fawcett and Collis-

George (1967).

The actual moisture content of the soil at the time of sample was measured by calculating the amount of water lost from a known weight of soil after oven-drying for 24 hours at 105°C.

Air, ground and soil temperatures were measured at each sample time. Values for wind, sunlight, and precipitation were obtained daily from the meteorlogical office at Silwood Park for the duration of the experiment. 42

SECTION 2: ISOLATION TECHNIQUES 43

2.1 MATERIALS AND METHODS

2.1.1 SAMPLING REGIME

Samples were removed from the field site at intervals and brought back to the laboratory for analysis. A total of twenty stakes, five of each of the four treatments, were removed. Of each group of five per treatment, all were used for Direct Observation (Section 3) whereas only three were used for Isolations. The number of samples was a compromise between statistical validity and analytical practicality.

Sampling followed a logarithmic time scale for the first month in order to obtain the greatest possible information of the early stages of colonization of the wood. Thus samples were taken at 2, 4,

8, 16, and 32 days for the untreated material, but only 16 and 32 days for the treated material. It was thought unnecessary to sample the treated stakes before sixteen days, which was substantiated by the results. Sampling of both untreated and treated material was then continued at three-monthly intervals covering 3, 6, 9, 12, 15 and 18 months.

2.1.2 ANALYSIS OF STAKES

2.1.2.1 PREPARATION OF SAMPLES

Each stake was carefully withdrawn from the soil and any adhering soil particles removed. Each stake was individually sealed in a separate plastic bag to prevent contact between samples. In the laboratory each stake was treated in exactly the same way.

All operations were performed on a Laminar Flow Bench Model

64T (Microflow Ltd., Fleet, Hampshire) and strict aseptic techniques employed at all times.

The surfaces of each stake were wiped clean with 95% industrial methylated spirit. Using a small, portable circular saw a 10mm thick slice was cut across the stake, 5mm above and 5mm below the ground—line.

The sealed radial faces were cut off first. This ground-line slice was 44

then cut radial longitudinally into two equal halves (blocks), the

direction of cut being from the sealed tangential face to the exposed

tangential face in order to minimise any carry over of micro-organisms

(Figure 2). The saw blade was sterilized by swabbing with alcohol be-

tween each cut. One block was immediately put into a labelled

Universal bottle containing Formalin-Acetic-Alcohol (FAA) and left to

one side to be used for light microscopy at a later date. The second

block was used for isolations and consequently was further treated

immediately retaining all aseptic precautions.

2.1.2.2 ISOLATION OF ORGANISMS

Each of the isolation blocks was cut tangentially into three segments using a sterile, single-edged razor blade to reveal faces at

3, 10, and 40mm from the exposed face. Using a Reichart Sliding

Sledge Microtome a section, 500pm thick, was cut at each depth. Each section was then divided into thirty-two equal portions (or splints), using a sterile scalpel. Each of these splints formed an inoculum particle to be incubated on selective media. The final size of these splints was 1.25 x 2.5 x 0.5mm. Initially eight splints were planted on each of four petri dishes containingnutrientmedium, using the scheme illustrated in Figure 3. The number was reduced to four per plate after the early samples since, with eight per plate, fungi growing from them rapidly merged together making separation more difficult.

One clear advantage of this method of obtaining inocula is the standardisation of the unit volume of wood sample, thus allowing more valid comparisons between treatments. Earlier techniques such as the saw-cut technique were criticized by Butcher (1971a) because of this lack of standardisation. The use of small inocula is also preferred since species are more often obtained in pure culture and a greater range of species encountered. 45

FIGURE 2 - Sampling Procedure

Stake removed from soil. Position of groundline marked in resin

Surfaces wiped clean with methanol to remove any adhering soil particles

1cm thick groundline veneer removed

Resin (r) removed from two sides. Resultant block cut in half

1. Microbial Isolation. 500 pm sections cut 2. Microscopic at 3,10 & 40 mm Examination from surface

r—, Half of groundline block fixed in FAA before sectioning for Direct Observations.

Each section further divided and incubated on selective media 46

FIGURE 3 - Isolation Procedure

Each section divided into 32 equal splints (inocula) using sterile scalpel

BI 1 CS1 B12 CS 2

BM1 Cl BM2 C2

CS3 813 C4 B14

C3 BM3 CS4 BM4

815 CS 5 BI 6 . CS6

BM5 C5 8M6 CS

CS 7 B17 CS8 B18

Cl BM7 C8 BM8

Sets of 4 splints placed onto the appropriately marked petri dish and incubated at 22°C

TT 4= 02 4 0 4= 02 4o n2

Bacterial-Inhibiting Cellulose Copper Sulphate Benomyl/Streptomycin Malt Agar (BI) Agar (C) Agar (CS) Agar (BM) 47

Due to constraints of time and the fact that successional

climax had virtually been reached by one year the isolation procedure was modified for the last two samples at 15 and 18 months. For stakes

sampled at these times initial incubation was in a damp chamber and not on nutrient media. A 5mm thick veneer was cut from the ground line of

each stake and incubated in a damp chamber (Figure 4). As fungi appeared on the cut surface of the veneer they were transferred to malt agar for purification and identification.

FIGURE 4 - Damp Chamber for Incubation of Groundline Veneers

, 1

r- - PLAN Groundline veneer

Plastic mesh I ~

SECTION ~~~~~~.~~~~~~~~~~~~I.--- Petri dish I s!veral layers Groundline of damp filter veneer Plastic paper (to main- mesh tain humidity) 48

2.1.2.3 SELECTIVE CULTURE MEDIA

Some preliminary experiments including a literature review

(see Booth, 1971a) were conducted to determine the most useful media and

at what concentrations their major constituents were to be used. As a

result of these preliminary tests and experience gained during the

course of the experiment the following four media were found to be of

greatest use, and used routinely (Clubbe and Levy,1977; Clubbe,1978,

1980) .

•A. Bacterial-Inhibiting Malt Agar, a broad spectrum medium

suitable for most fungi:

Malt Extract : 25g. Oxoid Agar 15g. NH4SCN 0.1g. NaN3 : 0.003g. *Streptomycin Sulphate : 1.0g. Distilled Water 1000m1.

*Streptomycin sulphate was added as a sterile solution after autoclaving since it is denatured by autoclaving. Streptomycin sulphate from a stock bottle was made up in sterile distilled water and then added to the medium immediately prior to pouring plates. However, it was found that the streptomycin stock became contaminated with fungal spores from the airspora which could then germinate and grow on the plates. To avoid this possibility the use of sterile 1g. vials of streptomycin sulphate was favoured. The streptomycin sulphate was dis- solved in a small amount of sterile distilled water which was introduced through the rubber seal with a hypodermic needle and then removed and added to the medium. The combination of streptomycin, ammonium thio- cyanate and sodium azide inhibited or killed all the bacteria isolated from wood thus preventing inocula becoming swamped by rapidly growing bacteria which could inhibit fungal growth from the splints by creating anaerobic conditions within the wood. 49

B. Cellulose Agar, based on Eggins and Pugh's medium (1962)

and consisting of:

Ammonium Sulphate : 0.5g. L-asparagine 0.5g. Potassium Dihydrogen Sulphate 1.0g. Potassium Chloride 0.5g. Magnesium Sulphate 0.2g. Calcium Chloride 0.1g. Yeast Extract 0.5g. Agar 20g. Ball-Milled Cellulose : 10g. Distilled Water to 1000m1.

The cellulose was prepared as a 4% suspension in distilled water of cellulose powder (Whatman Cellulose Power, CFI1, derived from cotton). The suspension was then ball-milled for 72 hours to reduce the fibre size. The suspension of ball-milled cellulose was then added to the other constituents of the medium after they had been steamed, but before autoclaving, all constituents then being autoclaved together.

Cellulolytic fungi isolated on this medium utilized the cellulose as a carbon source, thus clearing the medium. However, good clearance zones were usually only achieved with the small fibre size created by ball-milling. If the cellulose was not ball-milled very indistinct clearing was observed.

C. Copper Sulphate Agar was used to isolate copper tolerant species, usually associated with CCA treated timber.

Malt Extract : 25g. Agar 15g. *Copper Sulphate (CuSO4.5H20) 10g. Distilled Water 1000m1.

*The copper sulphate was autoclaved separately in a small amount of water and added to the other constituents after autoclaving. 50

D. Benomyl Agar as a selective medium for Basidiomycetes.

Malt Extract 12.5g. Agar 15g. *Benomyl 0.004g. active ingredient Streptomycin Sulphate 1.0g. (added as a sterile solution after autoclaving) Distilled Water 1000m1.

*Benomyl was obtained as Benlate which is a commercial pre-

paration of one part benomyl to one part inert carrier. For a final concentration of 0.004 g.1-I active ingredient, 0.008 g.l -1 of Benlate was added (Ben. 8). A stock solution of Benlate was prepared in 50% ethanol and the appropriate amount added to the autoclaved medium.

This stock solution was kept in a refrigerator for a maximum of two weeks.

All media were autoclave sterilized for 20 minutes at a pressure of 20 pounds per square inch (p.s.i.).

2.1.3 CULTURAL TECHNIQUES

After inoculation all the plates were incubated in the dark at 22°C, being brought into the light only for examination. The plates were viewed dairy and as each new fungus emerged from the inoculum it was subcultured onto a standard malt medium:-

Malt Extract 12.5g. Agar 15g. Distilled Water 1000m1.

Further subculturing was often necessary to separate dis- parate fungi growing together. This was particularly so when one fungus of a mixture was a prolific sporer with a rapid growth rate, such as Trichoderma viride. In such cases inoculation onto Czapek-

Dox medium facilitated separation since mycelial production and sporulation were reduced on this minimal medium. 51

Czapek-Dox Agar consisted of:

Sodium Nitrate 2.0g. Potassium Chloride : 0.5g. Magnesium Glycerophosphate: 0.5g. Ferrous Sulphate : 0.01g. Potassium Sulphate 0.35g. Sucrose : 30.0g. Agar 12.0g. Distilled Water 1000m1.

Any bacteria isolated were cultured on a Nutrient Agar:

'Lab-Lemco' Powder 1.0g. Yeast Extract : 2.0g. Peptone : 5.0g. Sodium Chloride : 5.0g. Agar : 20g. Distilled Water I000m1.

The p.H. was approximately 7.4. This alkaline p.H. dis- couraged the growth of fungi.

Once the isolates had been purified attempts were made to identify them. All identifications were based on sporing structures wherever possible (see Section 5 for full details).

2.1.4 Sectioning of Inoculum Splints

Once any fungi inhabiting the wooden splints had grown out onto the incubation medium and sufficient time had elapsed to allow for very slow growing species (up to 6 weeks) the splints were removed and the plates discarded. The inoculum splints were fixed in FAA and stored.

Thin sections, for light microscopy, were cut from representative splints using the sliding sledge microtome. Due to their small size the splints were supported in ice on the freezing attachment of the microtome whilst sections were cut. The sections were mounted in warm 1% safranin in glycerine jelly. As the stain cooled, the jelly set, forming a stable, temporary mountant,safranin was drawn into the section and lignified 52

tissue stained red. There were three reasons for cutting sections.

Firstly, to see whether any evidence could be found of the presence of

hyphae which could not be linked to those fungi actually isolated. In

this way estimates were made as to whether all fungi present had been

isolated. On some occasions it appeared that one fungus had been inhibited

by another. For example, rapidly growing and sporulating fungi such as

Penicillium spp. and Trichoderma viride were occasionally seen to prevent the emergence of a fungus with pigmented hyphae. However, this was observed only rarely. Secondly, any hyphal characteristics which could

be seen in the sections and which could be positively linked to an isolated fungus were noted for possible use during the Direct Observations.

Apart from the obvious characters of size and pigmentation of the hyphae and the presence of clamp connections, no really valuable characters were observed. Thirdly, and most significantly, the presence of any decay was noted. In this way the potentialities of fungi, particularly the cellulolytic stainers, to produce soft rot cavities under field conditions was noted. This provided good supporting evidence for assigning fungi to their most appropriate Ecological Group. 53

2.2 RESULTS

2.2.1 DESCRIPTION

2.2.1.1 2 DAY SAMPLES

Untreated Pine

After only two days exposure in the field both bacteria and fungi were isolated from within the wood.

At 3mm. bacteria were already present in large numbers in all three samples (see Appendix 2 for detailed lists). Although of several different genera, as exemplified by morphological characteristics such as colour, form and habit, they tended, on microscopical examination, to be predominantly Gram positive rods (Plates 4.1 & 4.2).

One inoculum showed bacterial growth at 10mm. whilst inoculum particles from 40mm. were all sterile.

Fungi were isolated from only one of the three stakes from which isolations were made and in this only from 3mm, subsamples from

10 and 40mm. showing no fungal growth. The fungi isolated were sparsely distributed and of several different species. Verticillium psalliotae was present in largest numbers and this in only three of a possible sixteen inocula. Two inocula produced Mucor plumbeus. Aphanocladium sp. and Epticoccum purpurascens each grew from two inocula. A more unusual inhabitant was found in three inocula. This was Xylaria sp., an ascomycete known to cause decay in wood, but usually associated with the later stages of attack.

Untreated Birch

Birch stakes, which were noticeably drier on sampling than were the pine, showed much less activity when small splints were incubated on nutrient media.

Some bacteria of the type found in the pine were isolated at

3mm. from two of the three sampled stakes, but in lesser numbers. 54

Only one fungal isolate was obtained from the birch and this

was Verticillium Zecanii which is a closely related species to V.

psalliotae isolated from pine.

All inocula from 10 and 40mm. were sterile.

2.2.1.2 4 DAY SAMPLES

Untreated Pine

Samples at four days exhibited a very similar pattern to that

shown at two days. Of the three stakes from which isolations were made,

one was totally sterile, one showed limited bacterial growth and only

the third sample showed both bacterial and fungal growth at 3mm, the

fungi being present in low numbers. V. Zecanii was again isolated.

The cosmopolitan stainer Aureobasidiwn pullulans was isolated for the

first time,being present in three inoculum particles. An unidentified

yeast was also present in two inocula.

Bacteria were isolated from five inocula at a depth of 10mm.

from the exposed face in one of the samples, notably the sample from

which fungi were present at 3mm.

All samples at 40mm, were sterile.

Untreated Birch

The birch samples showed a greater diversity than at two days,

probably associated with their increased moisture content. Bacteria were

present in all samples at 3mm. and in two at 10mm. V. Zecanii was iso-

lated from all three stakes and Fusarium avenaceum from one. One inoculum splint yielded the Arthrinium state of Apiospora montagnei and a further two showed the presence of the sap-stain Ceratocystis sp.

All inocula from 40mm. were found to be sterile.

2.2.1.3 8 DAY SAMPLES

Untreated Pine

After eight days exposure the distribution of micro-organisms was still very irregular. However, bacteria were present in quite 55

large numbers at 3mm, being isolated from one, thirteen and fourteen

out of a possible sixteen splints in each of the three samples, re-

spectively.

Only two fungi were present, both having been present earlier; ut A. pull!ans in three inocula from one stake, and Xylaria sp. in five

inocula from a second stake.

No organisms were isolated from 10 or 40mm.

Untreated Birch

The levels of colonization in the eight days samples were

essentially similar to those exhibited at four days with bacteria being

present in high numbers; in six, fourteen and sixteen inocula at 3mm.

and zero, eight and eight respectively at 10mm.

Fungi were still irregularly distributed. An unidentified

yeast was present in one stake, the second showed no fungal activity

whilst the third yielded four species of fungi. Two of these, V.

Zecanii and A. pullulans, had been previously isolated, but the other

two were new species to birch, although Aphanocladium sp. was isolated

earlier on pine. Torula herbarum, a stainer closely related to

Aureobasidium in distribution and habit was isolated for the first

time.

No fungi were isolated at 10mm. and no micro-organisms at

40mm.

2.2.1.4 16 DAY SAMPLES

Untreated Pine

Bacteria were present in very large numbers at 3mm. (8, 12,

16 in the three samples) although surprisingly in only one sample at

10mm.

Fungi seemed to be established in the surface layers of the wood with eight species having been isolated and some in large numbers. 56

They were still of the 'Mould' and `Stainer' type (see Section 2.2.2.).

In greatest numbers, particularly in one stake, was Trichoderma viride.

In two cases T. viride grew on both copper sulphate and benomyl media.

However, its growth was very disorganised and appressed, showing no

signs of sporulation and it could be classed neither as a copper tol-

erant nor a benomyl tolerant organism. However, it does exemplify the

ubiquitous nature of this important Hyphomycete which can survive in

most conditions. T. viride was also isolated at 10mm, from one stake,

illustrating its rapid growth rate. Mucor plumbeus was also widespread

and Fusarium avenaceum, F. sambucinum and Botrytis cinerea were also

present.

Epicoccum purpurascens was isolated twice from one stake and on thirteen occasions from a second and where it grew on cellulose medium was capable of clearing the cellulose. A second stainer was also isolated but this remained unidentified (Mycelia Sterilia Demat- iaceous 1 - IC/CC/MSD1).

With the exception of F. avenacewn these fungi were also isolated at 10mm, but in lesser numbers than at 3mm. In addition a second sterile stainer was isolated (IC/CC/MSD2).

No micro-organisms were present at 40mm.

Untreated Birch

The distribution of micro-organisms in the birch samples, in contrast to the pine, was still irregular probably associated with-their lower moisture content when compared to pine.

One of the three stakes showed no microbial activity. The other two samples showed evidence of bacterial colonization, Fusarium sambucinwn, the stainers A. pullulans and E. purpurascens and a sterile dematiaceous fungus. Two of the three samples showed the presence of

Aphanocladium sp. in one and two inocula respectively.

No micro-organisms were isolated at 10mm. or 40mm. 57

TREATED SAMPLES

Only after sixteen days exposure were CCA treated stakes

sampled. The delay in sampling treated material seemed to be justified

by the limited flora found in these samples.

Pine revealed bacteria in large numbers at both 3 and 10mm,

but a paucity of fungal colonists. These were an unidentified filamentous yeast in all three samples and two species of PenicilZiwn;

P. cyclopian in two samples and P. fennelliae in one. A. pullulans and

Mycelia Sterilia Hyaline 1 (IC/CC/MSH1) were both isolated in one case.

Two new stainers were isolated in small numbers from one sample. These were Phoma sp. A. and StytalzdiaW sp.

No fungi were present at 10mm. and no organisms at 40mm.

The treated birch stakes revealed a more limited flora than the pine, and were noticeably drier than all other samples. Two stakes were uncolonized, the third showing large numbers of bacteria at 3mm, but only three isolates of same fungus, Verticillium lecanii.

All inocula from 10mm. and 40mm. were sterile.

2.2.1.5 32 DAY SAMPLES

Untreated Pine

Bacteria were still present in very large numbers at 3mm. and to a lesser extent at 10mm.

A wide diversity of fungi was present at 3mm, numbering twelve species, some in quite high numbers. Mucor plutubeus and Fusarium avenaceum were the dominant components, whilst the stainer, Epicoccum purpurascens, was also present in large numbers. Other fungi present in lesser numbers were, Trichoderma viride, Penicillium cyclopian, Mucor hiemalis, Verticillium psalliotae, an unidentified filamentous yeast and an unidentified hyphomycete (IC/CC/MSH2). A. pullulans was present in quite high numbers and the stainer/soft rot fungus Drechslera dematioidea was present in one stake at both 3 and 10mm. growing, significantly, on benomyl agar. 58

At 10mm. from the exposed face bacteria were present, although

in low numbers and the dominant fungus was E. purpurascens which was

present in all three stakes. Four other fungi were irregularly dis-

tributed in two of the three stakes. These were M. pZumbeus, B. cinerea,

the Arthrinium state of Apiospora montagne and IC/CC/MSD1.

Untreated Birch

Birch stakes exhibited a similar range of species to the pine,

but in slightly lesser numbers. As in the pine E. purpurascens was the

dominant fungus, being present in all three stakes. A. pullulans was

also isolated in quite large numbers. Other fungi of limited occurrence were the unknown filamentous yeast, F. avenaceum and T. viride in one case. Also present were B. cinerea, V. psalliotae and IC/CC/MSH2.

Bacteria were also present in large numbers.

At 10mm. only bacteria were present, in low numbers, no fungi having penetrated the wood to this depth.

Treated Pine

In the treated pine the number of species was quite high by thirty-two days although none were dominant, their distribution being very localised.

Bacteria were present. Yeasts were present in higher concentrations than previously encountered. The range of fungi included two species of Penicillium, P. cyclopium and P. purpurescens. V. psalliotae was isolated from two stakes. E. purpurascens was isolated from all three stakes, although in only one case in each. Also present in low numbers was A. pullulans and an unknown dematiaceous fungus which failed to spore. Drechslera dematioidea was present in one stake.

At 10mm. bacteria were present and a single occurrence of a yeast and a sterile dematiaceous fungus. 59

Treated Birch

The level of colonization of the treated birch was still low

at thirty-two days. Few bacteria were present; 0, 2, 4 out of 16 in

the three stake sub-samples. Similar fungi occurred as had been shown

in pine; four cases of P. cyclopium and one of A. pullulans from one

stake, a single isolate of D. dematioidea from a second and no fungal

colonists from the third.

2.2.1.6 96 DAY SAMPLES

Untreated Samples

After three months exposure all untreated material was found

to be well above the fibre saturation point (@30% Moisture Content) in

the surface layers at the ground line and these outer layers were fully colonized. Fungi isolated exhibited their widest variety of species, many in quite high numbers. The types of fungi isolated were still mainly of the 'Mould' and 'Stainer' category, but for the first time soft rot fungi began to appear at significant levels and, in the case of birch, the first appearance of a basidiomycete.

Untreated Pine

Bacteria were still present at 3 and 10mm, although none were isolated at 40mm. from the surface of the wood.

Seventeen species of fungi were isolated at the 3mm. depth, showing the widest species diversity of the whole field trial.

T. viride, F. avenaceum, P. cyclopium, unidentified yeasts and IC/CC/MSH1 were again isolated. Fusarium sp. 1, which spored too sparsely to facilitate speciation was also isolated. The several new members of the flora were, Zygorhynchus heterogamus, a fungus allied to

Mucor, CyZindrocarpon destructans and PestaZotia sp. and a copper tolerant strain of Penicillium cyclopium. A final new member of the succession was Acremonium strictuur which was capable of clearing cellulose, 60

Various species in this genus, which has been the subject of a major

revision recently (Gams,197t), have been reported as having cellulases

and the capability of producing both erosion and cavities in wood in

culture (Henningsson and Nilsson, 1976).

A number of dematiaceous fungi were also isolated, although

in low numbers. E. purpurascens, the dominant stainer in previous

samples was found in only one case. Phoma sp. A and IC/CC/MSD4 which

was copper tolerant, were also isolated in low numbers. Three further

dematiaceous fungi were isolated and all showed soft rot capabilities.

All cleared cellulose rapidly when grown on cellulose medium. Of these

three, Phialophora fastigiata and Humicola fuscoatra, are fungi of

proven soft rot ability. The third remained unidentified as

IC/CC/MSD5, but its soft rot capabilities were indicated by the pos-

session of active cellulases, strong growth on copper sulphate medium

and the typical softening of the original inoculum splint caused by

visible soft rot cavities in culture.

The flora at 10mm. was still quite limited. Bacteria and

yeasts were found and several fungi, all of which had been isolated at

3mm. These were P. cyclopium, which cleared cellulose, T. viride and

IC/CC/MSH1. This latter unidentified fungus, which had a very slow

growth rate, was found to clear cellulose at a slow rate. However, it

did not respond to any methods of inducing sporing and, therefore, could

not be identified. A. pullulans was present in two of the three stakes, and a new stainer, Coniothyrium fuekelii, in one case.

Untreated Birch

Bacteria were still present at 3 and 10mm, although in smaller numbers, and three splints from one stake showed bacteria at 40mm. This was the first indication of microbial activity at 40mm. from the exposed surface in this wood species. 61

The fungal flora at 3mm. was manifestly different to that isolated at 32 days. Mould fungi were still present in high numbers as shown by the presence of yeasts, Z. heterogamus, T. viride, Pestalotia sp.,

B. cinerea, Fusarium sp. 1 and IC/CC/MSH3. However, the major change was the presence of Phialophora fastigiata in high numbers in two of the stakes and the presence of IC/CC/MSD5 in the third, both of these being copper tolerant, soft rot species not found previously. Also of note was the presence of the first basidiomycete, IC/CC/BS7. E. purpurascens, which cleared cellulose, was still present in high numbers and in two cases grew on the copper sulphate medium, although its growth was restricted. It has been reported as having the capacity to cause erosion damage in test material (Henningsson & Nilsson,197640.

The same basidiomycete, IC/CC/BS7, had reached 10mm. in one of the stakes, although the soft rot organisms had not. The distribution of the other fungi present,. A. pullulans, B. cinerea, Fusarium sp. 1 and an unknown yeast, was typically sparse.

Treated Material

The results for isolations from the three month material showed a dramatic change in the fungal composition to that revealed at one month. The appearance of Phialophora fastigiata in high numbers in both birch and pine in the surface layers was particularly significant.

Treated Pine

Bacteria were isolated irregularly at 3 and 10mm. Yeasts were isolated in quite high numbers at 3mm. and in one case at 10mm. P. cyclo— plum and V. psalliotae were found at 3mm. However, it was the stainer and stainer/soft rot fungi which were dominant at both 3 and 10mm. This represented a major change in the successional pattern. Most prevalent at both depths was Ph. fastigiata. IC/CC/MSD5 was also isolated at both depths. Humicola fuscoatra, Drechslera dematioidea, E. purpurascens and

ScytaZidium sp. were also present at 3mm. With the exception of Scytalidium 62

all these fungi were capable of growing on the copper sulphate medium,

although Epicoccum only to a limited extent, the other two being true

copper tolerant species. All of the fungi have been linked with the

decay of treated wood. All have cellulases and have shown erosion

activity in field and laboratory material (Henningsson & Nilsson,1976a;

Leightky,1978 + others). In addition Ph. fastigiata will produce

cavities as will Humicola grisea, a very close relative of H. fuscoatra.

The genus Humicola is currently undergoing revision and the presence of

a Glade rather than distinct species is being indicated (De Bertoldi,.

1976). Thus a similar functional role in the environment is likely for

H. fuscoatra. Other species of Humicola have also been reported as

causing soft rot (Seehan, Liese & Kess,1975).

Treated Birch

Preservative selection on species composition seemed to be the same in birch as that exhibited in the pine. The main difference was in

the rate of colonization and not in the species present. Thus Ph. fastigiata was still dominant at 3mm, but in lesser numbers when compared with the pine. H. fuscoatra, D. dematioidea and IC/CC/MSD5 were also present, but in similarly reduced numbers. Two additional stainers were also isolated in low numbers. These were Phoma sp. B and Cladosporium herbarum. Yeasts and P. cyclopium were also present at 3mm. The only fungus to have colonized to a depth of 10mm. was V. lecanii, and this in only one of the three samples, Verticillium again showing its pioneer role in colonization.

2.2.1.7 184 DAY SAMPLES

Untreated Pine

No species seemed to be dominant in samples harvested after six months. Ph. fastigiata, although still present appeared in only one sample at 3mm. and two at 10mm. The species diversity was large with some fungi represented in large numbers at both depths. 63

Six months revealed the first basiodiomycete in the untreated

pine. This was Sistotrema brinlananii, found at both 3 and 10mm. Al-

though a basidiomycete it showed no reaction on sawdust medium (see

Section 5) and its wood decay properties are, as yet, still unknown

(Baker et al.,1977). However, it has been linked to slow brown rot in

wooden window frames (Carey,1980). Its role as a possible parasite of

wood decay fungi has been discussed by von Aufsess (19760 and of

staining fungus in this study (see Plate 9).

Bacteria were isolated at 3 and 10mm. and, for the first time,

at 40mm. in two of the three samples.

Yeasts, F. avenaceum, M. hiemalis, P. cycZopium and IC/CC/MSH3

were isolated at 3 and 10mm. In addition Z. heterogamus and a sterile

mould were isolated at 3mm. P. cycZopium growing at 3mm. was copper

tolerant. These fast growing fungi were present in high numbers at both depths. Other stainers and stainer/soft rot fungi present were A. pullulans, the ascomycete fungus Coniochaeta subcorticalis, and three unknown dematiaceous fungi; the soft rot IC/CC/MSD5 and two stainers

IC/CC/MSDI and IC/CC/MSD6.

Untreated Birch

The untreated birch sampled at six months exhibited as wide a variety of fungal colonists, as was found at three months, but the dominant species were in a process of change such that basidiomycete fungi, which had barely colonized the wood at three months were now found to be well distributed in depth in all three samples.

Fqur species of Basidiomycetes were isolated from the surface layers of the wood (3mm.). Two of these were also found at 10 amd 40mm.

These were IC/CC/BS7, the basidiomycete isolated at six months which produced no decay when tested on sawdust medium (Section 5) and Stereum hirsutum which produced a strong white rot reaction. The two remaining species, found only at 3mm, were Sistotrema brinknanii and a second white rot, IC/CC/BS2. 64

The range of non-basidiomycetous fungi was large at 3mm. and correspondingly smaller at 10 and 40mm. The PhiaZophoras were still the most numerous fungi. Isolates from 40mm. were sufficiently different from the more normal Ph. fastigiata to be assigned a separate species,

Phialophora sp. 3. The soft rots D. dematioidea and IC/CC/MSD5 were present at 3mm. and the wood-decaying ascomycete XyZaria sp. was found at 10mm.

E. purpurascens and A. pullulans were still present in high numbers, now at all three depths. Other fungi present were T. viride,

V. psalliotae, F. avenaceun, Z. heterogamus, and Acremonium striatum at

3mm. Botrytis cinerea was isolated at 10 and 40mm. from one stake,

T. viride and F. avenaceum at 10mm, and A. stricture and an unknown mould at 40mm.

Typically, individual mould species were present in low numbers and randomly distributed, thus showing their functional role as an ecological grouping.

Treated Material

' A similar distributional pattern of fungi was exhibited in both wood species. The dominant fungus in both species at 3 and 10mm, and at 40mm. in one of the birch samples was Ph. fastigiata. A new soft rot fungus, Trichocladium opacum, was isolated at 3mm. from both pine and birch. The unidentified soft rot fungus IC/CC/MSD5 was present in increased numbers in pine at 3 and 10mm. and in birch at 3mm. D. dematioidea was present in low numbers at 3mm. in both species..'M other soft rot fungi were also found in birch at 3mm. These were H. fuscoatra and

Phialophora sp. 4, both from only one stake. IC/CC/MSD7 was isolated in one case from 10mm. in the birch. This fungus, which remained sterile and thus unidentified, had a very slow growth rate and cleared cellulose completely.

A single isolate of Cladosporium herbarum was obtained from pine at 3mm. 65

The 'mould' component was severely reduced in both wood species

and was represented by two species of Penicillium. These were P. cyclo-

pium and a new species, P. corylophilum present at all three depths in

the birch and at 3mm. only in the pine. F. avenaceum from 3mm, both

species of Verticillium from l0mm. in the pine, T. viride from 3mm. and

the copper tolerant strain of P. cyclopium from 10mm. in the birch com-

pleted the species list.

Thus the 'mould' and 'stainer' type fungi were being dis-

placed by the stainer/soft rot type fungi. These latter soft rot fungi

were represented by six different species, dominated by Ph. fastigiata.

2.2.1.8 265 DAY SAMPLES

Untreated Pine

The colonization after nine months exposure showed a pattern

essentially similar to that exhibited in the six month samples. Although

a major change in neither species composition nor dominance had occurred

colonization had progressed to a depth of 40mm. This was the first time

that any significant numbers of fungi had been found at this depth.

The microbial composition was similar to that revealed at 184

days. Bacteria were still present in large numbers at all three depths,

but now greatly reduced on the benomyl medium due to the addition of

bacteriocides. This medium was henceforth known as Benomyl/Streptomycin medium.

Yeasts were found at 3 and 10mm. T. viride was found scattered

throughout the samples at all three depths. P. cyclopium was isolated in

low numbers from 3 and 10mm, as was M. hiemalis, but in higher numbers.

F. avenaceum and Z. heterogamus were still present at 3mm. and an unknown

mould (IC/CC/MSH3) was present at 10 and 40mm, although in very low numbers.

The soft rot fungi Ph. fastigiata and H. fuscoatra were still dominant at all three depths. One isolate of D.dematioidea occurred at 66

10mm. on cellulose medium, which was cleared as the fungus grew. E. purpurascens, which similarly cleared cellulose, occurred at both 3 and

10mm, although in much smaller numbers than it had in earlier samples.

An unclassified dematiaceous fungus was also found on two splints at

3mm.

The basidiomycete S. brinkmanii was found in abundance at all three depths, being isolated each time on the benomyl/streptomycin medium. Previously it had been isolated at only 3 and 10mm.

Untreated Birch

By the nine month sample the Basidiomycetes had become established as the climax component of the microbial succession in untreated birch. The unknown basidiomycete IC/CC/BS7 and S. brinkmanii were present at all three depths. IC/CC/BS2 was isolated at 3 and 40mm.,

S. hirsutwn at 3 and 10mm. and IC/CC/BS13 at 10 and 40mm. The latter three Basidiomycetes were all white rots.

As a probable consequence of the increased importance of the

Basidiomycetes the presence of other fungi was severely reduced. Soft rot fungi were represented in only one stake and only in the surface layers by T. opacum and D. dematioidea. Phialophora was noticeable by its absence. E. purpurascens was still present at all three depths, in high numbers at 3mm., producing cellulases.

Of the mould fungi M. hiemalis was isolated at 3 and 10mm,

V. Zecanii at 3 and 40mm, F. avenaceum at 3mm. and B. cinerea at 10 and

40mm. These were all present in very low numbers and restricted in distribution. The notable exception was the re-appearance of T. viride in high numbers at all three depths and producing active cellulases.

Treated Material

In complete contrast to the untreated material, the treated samples revealed a dominance of soft rot organisms. 67

In the birch Ph. fastigiata and T. opaewn were isolated at all

three depths in large numbers and D. dematioidea was present in large

numbers at 10 and 40mm.

In the pine samples Ph. fastigiate was found at all three

depths, although still in quite small numbers at 40mm. H. fuscoatra was

present at 3 and 10mm. and the unknown soft rot, IC/CC/MSD5, at all three

depths. Phialophora sp. 4 was found at 3 and 10mm.

These soft rot fungi accounted for 75% of the total number of

isolates from birch and 50% of the total from pine.

The remaining fungi isolated were mainly of the mould type.

These were isolated in low numbers and in birch comprised A. pullulans

at 3 and 10mm, P. cyclopiwn at all three depths, V. psalliotae at 3 and

40mm, and B. cinerea and A. strictuur at 10mm.

Correlated with the lesser proportion of soft rots in the pine

when compared with the birch, the diversity of the rest of the myco-

flora was consequently greater in the pine. T. viride was isolated from all three depths, in surprisingly high numbers from 40mm. F. sambucinum was isolated from 3 and 10mm. as was the CyZindrocarpon state of Nectria coccinea and in addition Fusarium sp. 2 at 10mm. P. cyclopium was found at 3 and 40mm. and V. Zecanii at all three depths. A. pullulans was isolated at 10 and 40mm. and Scytalidisnr sp. at 3mm.

E. purpurascens was isolated at 10 and 40mm, A. strictuur at

10mm. and Gliocladium rosewn at 10mm. These three fungi all had active cellulase systems.

2.2.1.9 363 DAY SAMPLES

Untreated Pine

Even after one year's exposure the pine samples showed a wide species diversity, essentially similar to that of nine months, with no obvious dominants. The major advance on the previous samples, however, 68

was the presence, for the first time, of a second basidiomycete in

addition to S. brinlananii which had been a regular inhabitant for the

previous two samples and was again.present, at all three depths, in

this sample. The second basidiomycete, unlike Sistotrema, proved to be

a white rot fungus, but remained unidentified as IC/CC/B13.

Interestingly, it was restricted to the 40mm. depth, being present in

two of the samples.

Soft rot fungi were still in evidence as shown by the presence

of Ph. fastigiata and Ph. melinii at all three depths, although in very

limited numbers. An unknown soft rot, IC/CC/MSD9, was isolated on

copper sulphate medium at 3 and 10mm. Also present in large numbers at

3 and lOmm. was Coniochaeta subcorticalis. The presence of this

ascomycete was of great interest as this is one of the perfect stages of

Phialophora and may account for the paucity of the imperfect form.

ScytaZidiwn sp. and a pycnidial fungus, IC/CC/MSD8, were iso-

lated from a single stake each, and Phoma sp. A. was also present in one

stake at 3mm.

A variety of mould fungi were isolated at all three depths.

Gliocladiwn rosewn was isolated for the first time, being present in low

numbers at 3 and lOmm.

Other fungi present had been isolated before and comprised;

M. hiemaZis, F. avenaceum and P. cyclopiwn at 3mm, T. viride at 3 and

10mm. and four unclassified, sterile moulds.

Untreated Birch

The birch stakes sampled after one year showed the same basic pattern as that manifest in the previous sample. As at nine months, the basidiomycete fungi were dominant, together with T. viride in its role as a Secondary Mould, at all three depths. The Basidiomycetes were represented by seven species, two of which were new species to the 69

succession. Present at all three depths in relatively large numbers was

one of these new species. This was Bjerkandera adusta, a white rot

fungus. The second new species was also a white rot, but present at only

3mm. and as yet its identity is unknown (IC/CC/BS17). S. brinkmanii was

isolated at 3 and 10mm. and IC/CC/BS7 at all three depths. The other

Basidiomycetes isolated, all white rots, were S. hirsutum at 3 and 1Omm,

IC/CC/BS2 at 3 and 40mm. and IC/CC/BS13 at 1Omm,

A limited number of soft rot fungi persisted, irregularly

scattered through the samples. Ph. fastigiata was present at all three

depths, but in very low numbers and a single isolate of an unknown,

sterile soft rot (IC/CC/MSD9) occurred at 10mm.

E. purpurascens occurred in two of the stakes at 3mm. Iso-

lated at all three depths from one stake was the stainer Botryodiplodia theobromae.

The dominant member of the mould community was once again

T. viride. The secondary mould G. rosevun was also present at all three

depths, showing like Trichoderma, a characteristic clearing of cellulose

when grown on the ball—milled cellulose medium, indicative of active cellulase production. Others present were V. psalliotae and F. avenaceum at 3mm. and a sterile mould at 40mm.

Treated Pine

The flora present in treated pine samples was dominated by the soft rot type organisms. Apart from T. viride in one case at 3 and 1Omm, but in high numbers at 4Omm, and P. cyclopiwn at 3mm. in one stake, all other organisms isolated fell into the soft rot category. These numbered seven distinct species. Phialophora species were again dominant in terms of numbers of positive occurrences. Ph. fastigiata was present at 3 and

10mm, Ph. sp. 4 was present at all three depths, Ph. melinii at 3 and

10mm. and a new species, Ph. sp. 2,also at 3 and 10mm. H. fuscoatra was 70

again present in very high numbers at all three depths and T. opacwn was

active at all depths, although in lesser numbers. Finally, a single

isolate of an unknown soft rot, IC/CC/MSD5, was found at 10mm.

Treated Birch

The situation in treated birch after one year's exposure was

essentially similar to that in the pine, but with the levels of soft rot

colonization much greater in terms of number of isolates. Again the

Phialophoras seemed to be dominant with Ph. fastigiata and Ph. sp. 4 active at all depths, Ph. melinii at 10 amd 40mm and Ph. sp. 2 at 3 and

10mm. H. fuscoatra wa-s present in high numbers and T. opacum in lower numbers, both at all depths. D. dematioidea was again isolated at 10 and

40mm. and an unknown soft rot in one case at 40mm. Although this did not spore it possessed all the vegetative characteristics of a Phialophora species.

The appearance of large numbers of T. viride at 10 and 40mm. and to a lesser extent at 3mm. possibly indicated the availability of a new food source which could be exploited by the fungus. P. cyclopiwn was also scattered in depth through the samples.

2.2.1.10 500 AND 591 DAY SAMPLES

As described in Section 2.1.2.2., at the last two samples, fifteen and eighteen months respectively, a limited isolation procedure was adopted rather than the full—scale regime described for all previous samples. This involved incubation of 5mm. thick veneers of wood from the ground line (see Figure 4 ). Thus, only the organisms capable of living on the wood products and growing out of the cut end of the wood were identified, at the expense of other, more passive, inhabitants.

Several fungi were found sporing on the front (uncoated) face of some of the untreated stakes. These were the discomycete Phacidiwn sp. and the coelomycete Dinemasporium strigoswn. Both these fungi were quite 71

widespread. Also present in a sporing condition was TetrapZoa sp.,

again restricted to untreated stakes and also several unidentified species.

When the veneers were cut from the ground-line, visually they

fell into two categories. Those of the treated stakes exhibited a

characteristic staining throughout the depth of the wood. On incubation

the fungi which grew from the cut surface were of the soft rot type.

Trichocladium opacum spored directly on the wood in great profusion and

also a dark mycelial mat developed which, on subculturing onto malt

agar, proved to be of the PhtiaZophora type, mostly in the Ph. fastigiata

group. The findings were consistent in both wood species, thus re-

affirming the condition presented by the full isolation regime at one

year. However, only the birch showed soft rot attack indicated by the

presence of many cavities when sections were cut (see Section 3).

The untreated veneers, however, were manifestly different,

showing evidence of mainly basisiomycete presence, especially in the

birch. Presence of stain was limited in the birch, but still evident in

the pine.

Many of the 591 day untreated samples were matted with thick

sheets of the basidiomycete Phlebia merismoides which had not been iso-

lated previously. This new white rot fungus may have been replacing the

dominant decay fungi S. hirsutzan, B. adusta and IC/CC/BS13 of the one

year sample. Evidence for this replacement in the natural decay of hard-

wood stumps has been reported recently (Rayner and Todd, 1979). Thus,

the birch, by eighteen months, seemed to have reached a climax state

similar to that found in the decay of hardwoods in the natural environment. Pine samples still exhibited mostly IC/CC/BS13, but also some

P. merismoides. Both species of wood also showed the presence of another new basidiomycete, IC/CC/BS4. This remains unidentified, as yet, but was also a white rot. 72

Thus, the important fungi had been established by the full

isolation regime by one year and the two latter samples confirmed this

picture of soft rot dominance in the CCA-treated material and basidiomycete dominance in the untreated material. What does seem to be important in these later stages was the changing dominance of basidiomycete species in untreated stakes and the onset of decay in the untreated pine and the furtherance of decay in the treated birch. Treated pine stakes, although showing the same species composition as the treated birch were still immune from decay, even after eighteen months (see Section 3).

2.2.2 ECOLOGICAL GROUPS

In an attempt to quantify the successional nature of the colonization, all organisms isolated in position and time were assigned to the most appropriate "ecological group" (Clubbe,1980). Six groupings were constructed using two basic critera; observations during this study and results from similar studies published in the literature.

The six groupings thus created were Bacteria, Primary Moulds, Stainers,

Soft Rots, Basidiomycetes and Secondary Moulds.

The bacterial group included all the true bacteria which were mostly gram-positive rods, and the Actinomycetes such as Streptomyces sp., which showed a somewhat random distribution. Their presence was noted, but no real attempt was made either to identify them or to investigate them further (Plates 4.1 & 4.2).

The Primary Moulds were the first fungal colonists and are equivalent to Garrett's 'sugar fungi' (Garrett,1951, 1955). These included the yeasts. Since they possessed neither cellulases nor ligninolytic enzymes, and appear not to be capable of degrading the wood directly, their food source must have been soluble sugars and simple carbohydrates either present in the wood itself, principally the rays or axial parenchyma, (Plate 4.3-4.6) or derived from the soil. This food 73

PLATE 4

4.1 & 4.2 Bacterial smears stained using Gram stain.

4.1 Culture no. IC/CC/B19. Gram-positive rods of the Bacillus-type.

4.2 Culture no. IC/CC/B3. Gram-positive rods of the Actinomycete-type.

4.3 - 4.6 RLSs stained with Schulze solution (Chlor Zinc Iodine) to

show the presence of starch.

4.3 Untreated Scots pine showing ray cells packed with starch.

4.4 As 4.3, but showing the discrete nature of the starch grains.

4.5 Untreated birch showing starch grains (s) in the rays to be smaller

and less widespread than in pine. Most of the ray contents are

lipid material.

4.6 Untreated birch showing similar starch grains (s) in the axial

parenchyma. Most of the starch in birch is located in the paren-

chyma. • • .9

•

4.6 75

source must have been shared by the third group of organisms, the

Stainers, which closely followed the Primary Moulds, in successional

terms. These are non-decay fungi known for their discolouration of

timber due to their heavily pigmented walls (Findlay, 1959).

The fourth category were the Soft Rot fungi. Capable of break-

ing down the cellulose of the cell wall, they represented the first true decay fungi to penetrate the wood. They were of the stainer/soft rot

type, particularly the Phialophora group, characterised by the possession of pigmented hyphae, rather than of the non-pigmented Chaetomium

type so commonly used in experimental systems. Fungi were assigned to

this category on the basis of having caused observable cavities in the wood. Reference was also made to relevant literature for the known activities of the fungi isolated.

The basidiomycete group included all those fungi with clamp connections and those whose cultural characteristics and effect on sawdust distinguished them as Basidiomycetes.

The final group of organisms was the Secondary Moulds. This was a grouping to include all those 'mould fungi' which possessed an active cellulase system as exemplified by a clearance of cellulose in culture. Their position in the succession seemed to be associated with the appearance and eventual dominance of the decay fungi, particularly

the Basidiomycetes. As these fungi, predominantly T. viride and G. roseum, appeared to be unable to attack the wood itself, their role was• probably one of utilizing the cellulose, derived from the breakdown of the wood, which was in excess of the requirements of the decay fungi.

This cellulose food source may have been a true nutritional excess, or

the result of competition between the two groups of organisms for the

partially decayed wood.

Tables. 1-4 show a species list of all the isolates together with. their ecological status. It also indicates the position and time of occurrence of all fungi isolated.

76 TABLE 1 - Occurrence of Primary Moulds

ARTHRINIUM state of SPU >o BRU APIOSPORA MONTAGNE 3.- NNW _ 2 4 8 16 32 96 184265363 SPU 1g ...- BOTRYTIS CINEREA BR U ~.~--~ BRT t0 ... 2 4 8 16 32 96 184265363 265 SPU 3 CYLINDROCARPON DESTRUCTANS

SPT 3 CYLINDROCARPON state of 265 NECTRIA COCCINEA SPU T SPT 3 FUSARIUM AVENACEUM 184 BRU 2 4 8 16 32 96 184 265363 SPU 1g SPT I FUSARIUM SAMBUCINUM 265 BRU 3 2 4 8 16 32 96 184265363 . FUSARIUM SP.1 BR U 1g 2 4 8 16 32 96 184 265363 SPT 10 r. FUSARIUM SP.2 265

SPU 1 .MUCOR HIEMALIS BRU 1~ 2 4 8 16 32 96 184265363 SP U fs MUCOR PLUMBEUS al= 43 SPU ; ..~ SPT PENICILLIUM CYCLOPIUM BRU 3 BRT 4 2 4 8 16 32 96 184265363 16 32 96 184 265 363 SP U 3 PENICILLIUM FENNELLIAE SPU 3 _ PENICILLIUM PURPURESCENS 2 4 8 16 32 96 184 265 363 SPU 3 _ PESTALOTIA SP. BRU 3 — 2 4 8 16 32 96 184265 363 SPU — SPT'3 265 363 TRICHODERMA VIRIDE BRU 1~ 2 4 8 16 32 96 184 265363 SPT1s_ VERTICILLIUM LECANII BR U 3_ BR 3 16 32 96 184265363 SPU SPT VERTICILLIUM PSALLIOTAE BRU — — BRT 4; 2 4 8 16 32 96 184265363 16 32 96 184265363 SPT 10 Various YEASTS SPU I — 10 BRU 9 BRT 33 -- 32 96 184265363 16 32 96 18426 363 SPU 3 ZYGORHYNCHUS HETEROGAMUS BRU 2 4 8 16 32 96 184 265 363

SPU 3 NMI ME MYCELIA STERILIA BRU 3 .-. — 32 265

TABLE 2 — Occurrence of Stainers

SPU 3.. APHANOCLADIUM SP. BRU3 2 4 8 16 SP U SP T 'g AUREOBASIDIUM PULLULANS 16 32 265 BRU' BR T 13 1■I 11M1111= 4 8 16 32 96 184265 16 32 184265 BOTRYODIPLODIA THEOBROMAE BR Ul 363 SP 3 CERATOCYSTIS SP. 4

SPT 3 CLADOSPORIUM HERBARUM BR T 3 MIR 96 184

SP U 13 ••~ CONIOCHAETA SUBCORTICALIS* 265363

SP 1° - CONIOTHYRIUM FUCKELII* 96

SP 11 13 SPT' EPICOCCUM PURPURASCENS* 32 96 265 BR U4 2 16 32 96 184265363

SP 3 PHOMA FIMETI 96

SP U 3 MN SPT3 MEER 363 16 PHOMA SP.A

SP U3. 96 PHOMA SP.B BR T3 96

SP U'° MI SP T3 96 SCYTALIDIUM SP.* 363 16 265

TORULA HERBARUM BRU3 -8 SPU3.. - XYLARIA SP.* 2 8 BR U10. s 184 SP U13 ! ... SP T'3 MYCELIA STERILIA SP Ul BR 1.13 16 32 96 184 265363 •32- 184265 *Cellulolytic

TABLE 3 - Occurrence of Soft Rots

SP U 10, SPUR ~.. DRECHSLERA DEMATI0IDEA BRU 3 -- BRT' -~■ 32 184 265 32 96 184265363 SP U ;8 •- - SP T 96 265 HUMICOLA FUSCOATRA BRT;q 96 184 265363 S P U' '--' SP T' PHIALOPHORA FASTIGIATA BRU ;q BR T 96 184 265363 96 184265363500591 SPU' r SPT'3 A 363 PHI EIOPHORA MELINII BRT 4p1 . 363

SPT 3 PHIALOPHORA SPECIES 2 BR T13 ~t 363

PHIALOPHORA SPECIES 3 BRU" 363 SPT' PHIALOPHORA SPECIES 4 BRT - 184 265363

TRICHOCLADIUM OPACUM BRU 3 Iwo 265 184 265363 500 591 SP UT SP T ; IC/CC/MSD5 BRU 3 BRT 3 9a 96184 265363 S P U'3 MI IC/CC/MSD9 BR il 363

TABLE 4 — Occurrence of Basidiomycetes & SecDnc(ary Moulds

Non—Decay Basidiomycetes

SPU 1 SISTOTREHA BRINKMANII BRU 184 265 363

IC/cc/BS7 BR U ' 96 184 266 363

White Rots

BJERKANDERA ADUSTA BRU' 363 500 591 SP u ' a PHLEBIA MERISMOIDES BRU IN 591

SPU 3 MUN . STEREUM HIRSUTUM BR U 184 265363 500 591

IC/CC/BS2 43 BRU 184 265 363

IC/CC/BS4 SPU 3 •. BRU3 •• 591

SPU'§ 7 IC/CC/BS13 BR U' 265363500591

IC/CC/BS17 BRU 3 MEN 363

Secondary Moulds

SPU 3 MIN ACREMONIUM STRICTUM ..a BRU 4a BRT3 96 184 265

SPU' SPT3 Mown GLIOCLADIUM ROSEUM 265 BR U 363 sPU1 TRICHODERMA VIRIDE BRU' BRT3- 265 363 363 80

2.2.3 QUANTIFICATION

Once the status of all the isolates had been established, tables were constructed of the relative frequencies of the different ecological groups within the wood. This data is presented as Appendix 2.

The frequencies were calculated from the raw isolation data. Since the media used were selective for or against particular organisms or groups a relative frequency figure out of 32 was inappropriate. Although 32 samples were taken at each depth from each stake, even if present some organisms would not grow on certain media because of the selective additives.

Bacteria were capable of growing on both cellulose and benomyl agars giving them a maximum score of sixteen since eight samples were incubated on each medium. Although streptomycin sulphate was added to the benomyl agar (benomyl/streptomycin agar) during the study, this did not completely erradicate the bacteria, but severely reduced their growth, leaving them evident as a 'frill' around the splints, which from visual observation did not then appear to affect growth of any emerging fungi.

Primary Moulds and Stainers both grew on the bacterial-inhibiting malt agar, and cellulose agar and were given a maximum score of sixteen.

Soft Rot fungi grew equally well on the malt, cellulose and copper sulphate agars thus allowing them a maximum score of twenty-four.

Apart from an occasional species their growth was inhibited by the presence of benomyl in the benomyl/streptomycin medium.

The Basidiomycetes, although able to grow on malt, cellulose and benomyl/streptomycin media were often out-competed on the former two by the more rapidly growing Secondary Moulds and their presence was consequently masked due to the rapid sporulation of the Secondary Moulds.

Therefore, to avoid the possible underestimation of their importance, only

their occurrence on benomyl/streptomycin was counted, giving a maximum

score of eight. 81

The Secondary Moulds grew on malt and cellulose and could thus attain a maximum score of sixteen to give a relative frequency of 100%.

Thus, the frequencies were calculated on the basis of a maximum score of

16 for Bacteria, Primary Moulds, Stainers and Secondary Moulds, 24 for

Soft Rots and 8 for Basidiomycetes.

This raw data of relative occurrence was then fed into the computer. Relative frequencies were calculated for each set of data

(see Appendix 2) and a surface was constructed which presented in graphical form the relative frequencies with time of each of the six major groups in the four different treatments. These results are presented in the following section.

In the following sections the terms relative frequency or frequency of isolation refer to the relative occurrence defined as

Positive Isolations x 100% and calculated in the manner explained above. Total Possible

2.2.3.1 UNTREATED BIRCH

The results for the relative frequency of isolation of the six groupings in untreated birch samples are presented in graphical form in

Figures 5, 6 and 7.

Several points are evident from the 3mm. graph (Figure 5).

Bacteria rapidly reached a peak at 8 days, showing a frequency of isolation of 75% and gradually fell away to zero at 363 days. Primary

Moulds, having colonized by 2 days, fluctuated, reaching a peak of 48% at 96 days and fell away to a very low level of 2.1% at 363 days.

Stainers followed a similar pattern of early colonization, fluctuation, peaking at 48% at 32 days and dropping to 7.1% at 363 days. Soft rot fungi appeared at 32 days, showed a slight increase and fell away to very low levels at 363 days. The Basidiomycetes rapidly increased in frequency of isolation once they had first colonized the wood at 96 days. 82

FIGURES 5-16. The Succession of Organisms, as Ecological Groups, on Wooden Posts

Key to Ecological Groups

B - Bacteria PM - Primary Moulds S - Stainers SR - Soft Rots BS - Basidiomycetes SM - Secondary Moulds

Figure 5 : Untreated Birch - 3mm depth

Figure 6 . Untreated Birch - 10mm depth

Figure 7 Untreated Birch - 40mm depth

Figure 8 : Treated Birch - 3mm depth

Figure 9 : Treated Birch - 10mm depth

Figure 10 : Treated Birch - 40mm depth

Figure 11 : Untreated_Pine - 3mm depth

Figure 12 : Untreated Pine - 10mm depth

Figure 13 : Untreated Pine - 40mm depth

Figure 14 : Treated Pine - 3mm depth

Figure 15 : Treated Pine - 10mm depth

Figure 16 : Treated Pine - 40mm depth

FIGURES 5-7: Untreated Birch

Fig. 5: 3mm t0° 50' of 60'

F REST*~ 4°`

4,4, v 4- ° mi ~ 3~ 67 a ~` cf4 OJ Q

Fig. 6: IOmm too so'

Of 60.' de a3 40! 15°~~~.1 20~

IsPir 40 Otr 4.4. Q. O €0 001 e• Co c2 e e'., csQ, 5 0 ..), Q

Fig. 7: 40mm

f 60. o JO" co4 aj.

,0.

eto t6- rib Q.`0 0 esQ h c.~ Y c2"'s CS '-s— • 0~ 84

Secondary Moulds, although not present at 96 days, showed a similar rise

in frequency once they were established at 184 days.

The predominant feature of the 10 and 40mm. depths (Figures 6

and 7) is the gradual increase in frequency of basidiomycete fungi with

Secondary Moulds showing a similar rise. The other groups, including the

Soft Rots, occurred in much smaller numbers, reflecting a more erratic

distribution and showing their inability to fully colonize in depth.

At 10mm, Bacteria appeared at 4 days and much later at 40mm,

showing no signs of colonization until 96 days. Fungi, as indicated, also appeared much later; at 96 days at 10mm. and 184 days at 40mm.

This lag period was manifest in all samples. However, the one exception to this was the Basidiomycetes which appeared at about the same time at all depths.

2.2.3.2 TREATED BIRCH

Figures 8, 9 and 10 illustrate, graphically, the relative frequency of isolation of the six groups in treated birch.

Soft rot fungi, shown at the front of the graph, showed a marked increase with time, reaching 94.4% at 3mm, 68% at 10mm. and 44.4% at 40mm. after 363 days exposure. Secondary Moulds were the only other group to reach a high relative occurrence and this only deeper in the wood. Primary Moulds were consistently present, although in low frequencies, and reached a maximum at 184 days. Stainers were also present, but at very low frequencies, reaching a maximum of 18.75% at

265 days, but never reaching 40mm. Bacteria fluctuated greatly, but never showed very high frequencies and barely colonized 40mm. (6.25% at 184 days only).

No Basidiomycetes were isolated at any time

2.2.3.3 UNTREATED PINE

The results for the untreated pine stakes are presented in

Figures 11, 12 and 13. 85

FIGURES 8-10: Treated Birch

Fig. 8: 3nnn ,00

Fig. 9: IOmm 100

of 60 Ci r4 0t4 Ft'O..$ A11 4° f ~so"~.,.l ,.0

Fig. 10: 40nnn ,00 86

FIGURES 11-13: Untreated Pine

Fig. 11: 3mm

of 60 ,o+~ 4 F RESS0 *

o„ 9 0(5

~� ' •'? • ~Q. 4- Jo

Fig. 12: 10mm to° 50"

of 60. mes,

10"

str

Fig. 1 3: 40mm 10° so' 0" of 6 C FRESSo ce, 1 10'

Q Ov y 87

Generally there was a high level of colonization. The wood was colonized to a depth of 3mm. very quickly. Even by 2 days 54% of samples showed evidence of bacterial colonization, 25% showed Primary

Moulds and 18.75% Stainers. Bacteria and Primary Moulds, although fluctuating, were present in relatively high numbers throughout, peaking at 93.75% and 97.9% respectively at 184 days. This picture was repeated at 10mm, but with all values slightly lower and Primary Moulds not appearing until 16 days.

Stainers were also present throughout. They peaked at 52% at

32 days, dropped to 12.5% at 184 days, but picked up to 25% at 363 days.

At 10mm. the Stainers reflected a similar pattern, but did not appear until 16 days. However, their relative frequencies tended to be a little higher, still showing a frequency of isolation of 54% at 363 days.

The soft rot fungi never became important, in terms of numbers, since they reached a maximum of only 15% at 96 days and remained at about this level throughout. At 10mm. the Soft Rots exhibited similarly low frequencies.

Basidiomycetes appeared only after 184 days and then at 3 and

10mm. at 54% and 48.8% respectively. They showed a gradual increase in frequency of isolation up to 100% at 3mm. and 58.3% at 10mm. in the 363 day samples. At 40mm. they appeared slightly later, showing frequencies of 29% and 33.3% at 265 and 363 days respectively.

Figure 13 shows the frequency of isolation of all groupings at

40mm. No fungi appeared until 265 days, only a small number of Bacteria having been isolated at the previous sample. Primary Moulds, Bacteria and

Soft Rots appeared at the same time and were still present, at 363 days.

Some Stainers and Secondary Moulds also appeared at 363 days.

2.2.3.4 TREATED PINE

Represented in Figures 14, 15 and I6 are the results for frequency of isolation of all groups from treated pine. 88

FIGURES 14-16: Treated Pine

Fig. 14: 3mm

Fig. 15: IOnnn foO

0 0 1 6 .$,r4d . F%£o A17~ 4° ~ ~#'l.,.1 2.0

Fig. 16: 40nnn 89

Bacteria, Primary Moulds and Stainers had all colonized to a

depth of 3mm. by the first sample at 16 days. Bacteria had already

reached 100% and remained throughout at an average frequency of 62%.

Primary Moulds gradually increased from 20.8% up to 75% at 184 days and

then fell away to 6.25% at 363 days. Stainers, although present until

265 days never exceeded 12.5%. Soft Rots appeared in very low numbers

at 32 days, but rapidly reached 65.3% by the next sample and continued

to increase to 91% at 363 days. The last two samples showed the appear-

ance of some Secondary Moulds at 6.25% and 10.4% respectively.

The situation at 10mm. (Figure 15) revealed quite a different

picture. Bacteria remained relatively abundant throughout, averaging

55% for the first year with a range between 25% and 100%. All the

fungi, with the exception of the Soft Rots, failed to make any impact

in terms of numbers. Except for a frequency of 66.7% for the Primary

Moulds at 265 days all three groups, Primary Moulds, Stainers and

Secondary Moulds, never exceeded a low 16.7% frequency of isolation.

However, as exhibited at 3mm. the soft rot fungi once present gradually increased to a maximum 86% at the last sample.

At 40mm. (Figure 16) the soft rot fungi were beginning to get established. Having appeared at 265 days at 7%, they had increased to

27.7% by the following sample at 363 days.

One noticeable factor was the very large numbers of Primary

Moulds, all Trichoderma viride, at both 265 and 363 days. Stainers were isolated only at 265 days but at only 14.6% frequency. Bacteria were found at 184 and 265 days, but in very low numbers, 8% and 12% respectively. 90

2.3 DISCUSSION

From the results presented in this section the fungi colonizing

wood in the soil can be divided into Functional or Ecological Groups.

Assignment of fungi to the appropriate group was based on physiological

and ecological properties. These Ecological Groups ignore taxonomic

boundaries and it is only by recognising these divisions that the funda-

mental nature of the colonization process can be fully understood. Some

fungi fell between two groups, the siting of Trichoderma in the later

stages of colonization being particularly difficult. However, the

majority were assigned to the appropriate category with relative ease.

Once the organisms were grouped in this way an organised succession of

the wood was observed rather than disorganised colonization by diverse

organisms. By approaching the problem in this way it is then possible

to demonstrate substrate relationships and show how different substrates

affect the nature of the succession.

In the present trials the first colonists were always Bacteria,

either exclusively, or together with some Primary Moulds. The bacterial

group, although of a relatively diverse nature (Plates 4.1 & 4.2) were

predominantly composed of several species of Bacillus. The bacilli, apart from one species, are mobile by means of flagellae

(Bergey, 1974). Many of the bacteria colonizing wood produce pectinolases and cellulases (Gyllenburg & Eklund,1974). Thus, colonizing bacteria

have the capacity for penetrating the wood and attacking the pectin of

the pit membranes. It would seem likely that the bacteria were carried

in with the water front, but also capable of some directional, flagellar generated, movement. Adherence to pit membranes would afford a stable

support from which to attack the pectin. Destruction of the membranes

would allow passage of both water and organisms to neighbouring cells,

Primary Moulds, which were chiefly. Hyphomycetes and Zygomycetes, could 91

then enter the wood via these extra holes, as well as through the natural

radial pathways of the ray cells. Thus, in the early stages of coloni-

zation the natural pathways through the wood were exploited by a variety

of fungi with bacteria creating some extra holes. However, none of

these early colonists altered the wood to any appreciable extent, either

physically or chemically,apart from the presence of themselves, and

their metabolic products.

In successional terms Bacteria were isolated at the same time

as the Primary Moulds at 3mm, but in greater numbers, although quanti-

tative comparisons between unicellular rods, 1-7.pm long, and a

three-dimensional mycelial network many centimetres in diameter present

their own difficulties. However, at 10 and 40mm. Bacteria were consist-

ently isolated before Primary Moulds. The exception was the treated

birch where bacterial and Primary Mould components were isolated

concomitantly, probably due to the much slower uptake of water.

Although wood is primarily lignin and cellulose the ray cells,

even after kiln drying and preservative treatment, contain simple

carbohydrates and sugars (see Plates 4.3 - 4.6), Being unable to attack

the wood directly the Primary Moulds must have derived their energy from

these sources and possibly from the Bacteria. Bacteria isolated from

decaying wood have been shown to fix atmospheric nitrogen (Seidler et al.,

1972; Aho et al., 1974; Baines & Millbank,1976) and this could be made

available to the Primary Moulds as a result of bacterial death.

Bacteria may also supply essential vitamins or growth promoting sub-

stances (Shigo,1965; Henningsson, 1967b).

Following rapidly behind the Primary Moulds were the Staining

Fungi. These were primarily Dematiaceous Hyphomycetes, but also included some Ascomycetes and Coelomycetes. Their unifying factors were their pigmented hyphae and method of colonizing the wood. Although entering 92

the wood along the rays they rapidly penetrated the axial elements

(fibres or tracheids) passing from cell to cell by first forming a trans-

pressorium as a response to the hyphal tip coming in contact with the

cell wall. A fine penetration hypha was produced which mechanically or

enzymatically, or a combination of both, passed through the cell wall.

Once through the wall the hypha resumed its original diameter (Plates

5.1 - 5.3). It is thought that the stainers derive no nutritional

benefit from the wood itself and like the Primary Moulds are merely

scavengers. of simple organic compounds. Stainers reached their peak at

32 days at 3mm, after which time they colonized the deeper portions of

the wood, disappearing from the surface. Their whole growth pattern

seemed to indicate a role as an ephemeral scavenger. Treatment with

CCA was particularly effective against staining fungi which were

virtually eliminated from both the treated birch and treated pine. This

may be due to loss of soluble nutrients as a result of preservative

treatment, since the number of Primary. Moulds was also reduced.

The most likely food source for all the early non-decay

colonists is the reserve starch in the ray parenchyma of both wood species

and the axial parenchyma of birch (Plates 4.3 - 4.6). Starch grains are

easily broken down to glucose by the amylases of these early colonists

and absorbed into the mycelium (Cochrane, 1958; Perlman, 1965; Burnett,

1976). Differences in the early levels of colonization of the two wood

species by non-decay fungi may be attributed in part to the distribution

of starch grains. The presence of starch grains is very widespread in

the ray parenchyma of untreated pine (Plates 4.3 & 4.4). This must form

a readily accessible carbon source for the early colonists and due to

the wide distribution of rays containing starch must provide a reasonable volume of food. In comparison starch grains in the ray parenchyma of untreated birch (Plate 4.5) are much smaller, fewer in number and less evenly distributed. The bulk of the starch grains are located in the 93

axial parenchyma (Plate 4.6) which are relatively less accessible than

the rays. The preservative treatment and leaching schedule reduced the

amount of starch in the rays thus reducing the volume of food for the

early colonists. Thus superimposed on the slower uptake of water in the

birch is the availability of less food material in the form of accessible

starch. Both these factors contribute to the lower numbers of non-decay

Primary Moulds and Stainers in the birch when compared to the pine.

Although principally living on soluble material in the ray and axial parenchyma extra nitrogen may have been provided by the 'wick effect' (Baines & Levy, 1979). Uju (1979) showed that if soluble nitrogen was added to sterile soil containing half-buried sterile stakes of wood, the wood picked up this added nitrogen. The stakes showed a significant increase in nitrogen in both the below-ground and above-ground portions. Baines and Levy (1979) have attributed this to wick action.

If the stakes were colonized by micro-organisms they could lock up this nitrogen as biomass at the groundline preventing much of the transported nitrogen being deposited at the top of the stake. Uju (1979) showed this to be the case using stakes in unsterilized soil. Some stakes had the groundline area sealed using an epoxy resin whilst others were left open to colonization. In those stakes with the groundline sealed there was an increase in nitrogen in all portions of the stakes, above, below and at the groundline with a concentration gradient from below-ground to above- ground portions of the stakes. In unsealed stakes the greatest increase was at the groundline, much of it due to biomass nitrogen. The increase in the above-ground portion could not be attributed to biomass nitrogen.

Therefore, much of the transported nitrogen must have been assimilated at the groundline by the micro-organisms whilst a significant amount was deposited above-ground by wick action. Ofori (1977) showed a similar distribution of calcium in exposed CCA-treated beech fence posts which was not seen in unexposed posts. His results can be interpreted as being the 94

result of wick action. Thus, it is likely that any soluble nutrients in

the soil could be transported by wick action to a sink at the groundline

where colonizing micro-organisms could utilize them.

The first decay fungi in the succession were the Soft Rots.

These soft rot fungi were capable of attacking the cellulose and hemi-

cellulose of the wood cell wall and were, therefore, not dependent upon

simple carbon sources as were the earlier colonists. They first appeared

in low numbers after thirty-two days and gradually colonized the whole

block.

In the treated material the Soft Rots became well established

in the surface zone between thirty-two and ninety-six days and gradually

increased in numbers, at the same time beginning to colonize in depth

in large numbers. The actual species composition was the same in both

birch and pine, but as revealed by the Direct Observations (Section 3)

no decay was observable in the treated pine. Therefore, although de-

riving energy directly from the wood in birch, the Soft Rots were exist-

ing in the treated pine without attacking the cellulose. Therefore, the

question of what is their primary food source in this case needs to be

answered. This problem is discussed in Section 4.

In the untreated material the Soft Rots never got established,

being displaced very quickly by the rapidly growing Basidiomycetes. Peak

soft rot occurrence in birch (184 days) corresponded with the first appearance of Basidiomycetes in high numbers after which time the Soft Rots declined and the Basidiomycetes became dominant. A similar situation occurred in pine, although the Soft Rots did not disappear as quickly.

Basidiomycetes were not isolated from treated birch nor treated pine at any time, whereas after an initial lag period of between three and six months they rapidly colonized the whole of the untreated stakes.

Thus, by six months Basidiomycetes were present at all three depths in birch and at 3 and 10mm. in pine. In all subsequent samples Basidiomycetes 95

were isolated at all three depths in both species.

The distribution patterns of the fungi, particularly the

Basidiomycetes, during the first six months was very illuminating.

From being absent at three months to almost total colonization of all

untreated stakes at six months represented a very characteristic be-

haviour. In the complex environment of the soil it would be unlikely

that a single factor would govern this behaviour, accounting for the

initial lag period, rather than several interacting factors.

Species isolated as Primary Moulds, particularly Mucor, Fusar- ium and Penicillium, are amongst the most widespread in the soil and usually present as spores. Basidiomycetes, however, are present in the soil principally as hyphae (Warcup, 1951; 1957). Growth rates of Basidi- omycetes through the soil are generally much slower than those of this mould group, despite the fact that Basidiomycetes may already be present as actively growing hyphae (Burges, 1960). This difference in growth rate was also reflected in cultural studies of fungi isolated during the present study. Thus it would seem that the early colonists can germinate very rapidly and colonize the wood to exploit any soluble material. Few mucoraceous fungi can utilize carbon sources more complex than simple sugar (Jefferys at al., 1953). This, however, is compensated for by their rapid germination and colonization of material in precedence over other soil fungi. Growth rate differences alone would not account for the late appearance of the Basidiomycetes. Other early colonists may have a fungistatic effect. Trichoderma viride, for example, can produce the toxic metabolites gliotoxin and viridin which cause lytic changes in other fungal mycelia (Weindling, 1934, 1941). Bacteria can also cause lysis of fungal hyphae. Park (1956) found that Bacillus marcerans isolated from soil would actively lyse fungi in vitro, apparently by production of extracellular lytic material. Bacteria isolated during this study have shown some inhibitory effects towards the fungi. Stevenson (1956) found 96

that Streptomyces antibioticus lysed the hyphomycete Helmin4thosporium

sativzum in mixed culture in the soil, the lytic action being a combin-

ation of actinomycin and some other unidentified factor. Other examples

pervade the literature. It is interesting to note that the Zygomycotina

as a group do not produce antibiotics and disintegration of their hyphae

by bacteria is often recorded. The long-lived mycelium of Basidiomycetes,

many of which produce antibiotics, seldom appear to be attacked by bacteria

in the soil (Burges, 1960).

Thus a combination of slower growth rates, fungistatic action

and possibly time- for induction of cellulases and ligninolytic enzymes

results in a lag of three months before substrate colonization by Basidio-

mycetes. Induction time for wood-destroying enzymes may be a vital factor.

Siu (1951) proposed that it was the production of suitable cellulases that

probably delayed colonization of substrates containing cellulose by

cellulose-degrading fungi.

The final group of organisms associated with the colonization of

wood was the Secondary Moulds which were associated with the climax organisms; Basidiomycetes in untreated and Soft Rots in treated wood. The clearance of ball-milled cellulose in culture by these fungi showed their possession of an active cellulase enzyme system. Thus, although capable of utilizing cellulose as a carbon source, they seemed unable to attack the wood directly. They must have derived their energy from more simpler sources which most plausibly were the breakdown products of the decay fungi. This may have been a direct competition for breakdown products or more likely a 'mopping-up' process since they tended to occur after the decay fungi, particularly in the untreated birch. They occurred in lower numbers in the untreated and treated birch, and there was little evidence of them in treated pine.

The differing moisture contents of the four types of stakes was of interest, particularly the rapid, early wetting of treated pine stakes. 97

Baines (1980) has shown in field stakes that treated samples were con-

sistently drier than untreated ones and that no real difference was found

between species, that is, between untreated pine and birch or between

treated pine and birch. Baines's stakes were smaller (2cm2, cross—section)

than those used in this study (3 x 4.5cm), were unsealed and treated with

a commercial loading, a 3.5% solution of CCA, giving approximate dry salt -3 retentions of 26 kg m . This could indicate the importance of stake size

and concentration of preservative, the lower concentration being less water repellant. The larger dimension stakes used in the present study did not seem to dry out significantly once they had become wet, due to the presence of only one evaporative face, the 3cm unsealed, tangential face.

A relatively stable environment was thus provided for the organisms.

A summary of the colonization data is presented in Figures 17,

18, 19 & 20. This indicates the successional nature of the colonization showing relative occurrences of all groups at the three sampling depths in all four treatments.

This discussion is based on the generalities which can be drawn from the collected data, outlining overall trends. Specific comparisons are more difficult and their interpretation more conjectural. The role of Bacteria in the two species is possibly different since their numbers were consistently greater in the pine than in the birch, regardless of treatment, throughout the first year of exposure, although the morphological characters and species composition were comparable. Higher numbers in pine are more likely to reflect its higher moisture content at the time of sampling.

The assignment of T. viride to its most appropriate functional group was difficult since in the early stages it acted as a Primary Mould and showed no evidence of active cellulases in culture. However, at the later stages, especially those strains from the untreated birch, cellulase 98

production was clearly indicated in culture and thus it was probably functioning as a Secondary Mould. This may be indirect evidence for the description of T. viride as a form genus and its division into distinct species as suggested by Rifai (1969) and later adopted by Dennis and

Webster (1971a,b). More detailed morphological examination of the isolates may have split the Trichoderma isolates into different species, each with its own role in the succession, as a Primary or Secondary Mould.

Alternatively, the cellulases may be inducible enzymes and only activated by the presence of available cellulose as in the later stages in the breakdown of wood by the decay-fungi. This latter hypothesis would fit the distribution data of large numbers of Secondary Moulds associated with the Basidiomycetes in untreated birch.

The whole tenor of this discussion is based purely in isolations of micro-organisms from wood growing on nutrient rich, agar based media. It can only be assumed that this reflected the actual state of the organisms within the wood. As has been discussed earlier no distinction between the nature of the original propagule within the wood can be made, whether active mycelium, moribund mycelium or dormant spore. These problems are further discussed in the light of the results from the Direct

Observations in Section 4. 99

FIGURES 17-20. Summary of the Colonization Data for the First Year of the Succession

Key to Ecological Groups

B - Bacteria PM - Primary Moulds S - Stainers SR - Soft Rots BS - Basidiomycetes SM - Secondary Moulds

Figure 17 - Untreated Birch - 3, 10 & 40mm depths

Figure 18 - Untreated Pine - 3, 10 & 40mm depths

Figure 19 - Treated Birch - 3, 10 & 40mm depths

Figure 20 - Treated Pine - 3, 10 & 40mm depths

In all cases the height of the bars comprising the histograms are proportional to the Percentage

Frequency of Isolation (1mm = 10%)

100

FIGURE 19 - TREATED BIRCH FIGURE 17 = UNTREATED BIRCH

Ecological Group B PM S SR BS SM Ecological Group B PM S SR BS SM Depth Imm) 3 1040 3 10 40 3 1040 3 1040 3 10 40 3 1040 Depth (mm) 3 1040 3 1040 3 1040 3 10 40 3 1040 3 1040

363 MI 1111. 363 II —11111. 265 _ I.. Eli mil 265 = Mimi — 11111

184 ■,..a ■AMO ■mai AIM T I M E 184 _ am 96 II _ ` ■ _■ ( days) 96 L_ — — ■ TIME 32 ILI — ■ 32 ; --— _ (days) 16 II — N 16 ■ -

8 1111 - 111. 4 K — — 2 u

FIGURE 18 - UNTREATED PINE FIGURE 20 TREATED PINE

PM S SR BS SM Ecological Group B PM S SR BS SM Ecological Group B Depth (mm) 3 10 40 3 10 40 3 10 40 3 1040 3 1040 3 10 40 Depth (mm) 3 1040 3 1040 3 1040 3 10 40 3 10 40 3 1040

363 r~ ~■■._ 363 IIILI - 265 NEW MN - 265 W loll — 184 1111L 11. - TIME 184 IL_ IL.

96 BM La NMI (days) 96 ` L — ILI TIME 32 ■... 32 ■_ (days) 16 IL IL 16 m mu 8 ■ — 4 101

SECTION 3: DIRECT OBSERVATION TECHNIQUES 102

3.1 MATERIALS AND METHODS

3.1.1 PREPARATION OF MATERIAL

The sampling procedure from field site to laboratory has

already been described in detail in Section 2.1.1. From each sample

stake the groundline area was aseptically removed in the laboratory.

This comprised a block one centimetre thick to include half a centimetre

either side of the marked groundline. The resin which had coated three

sides of the stakes to prevent entry of organisms, thus allowing uni-

directional penetration through the uncoated outer tangential face, was

removed from the two radial faces of the groundline block (see Figure 2,

Section 2.1.2.). This block was then sawn in half and one half used

immediately for isolations (see Section 2.1.2.) whilst the other half was put in Formalin-Acetic-Alcohol (FAA). These latter halves formed

the raw material for all the direct observations. The formulation of FAA used was:-

Formaldehyde 5 volumes Glacial Acetic Acid : 5 volumes 70% Ethanol : 90 volumes

After complete infusion with FAA radial longitudinal sections (RLS) were cut from these half-blocks using a Reichert Sliding Sledge Microtome

(Figure 21). Although complete radial sections could be cut, due to their length (45 - 50 mm.) tearing of the cell walls was evident when viewed under the microscope. Consequently, complete sections were cut only for preliminary observations. Before the sections which were to be scored and fully analysed were cut the block was sawn in half in a tangential direction (see Figure 21). Thus the sections werē•cut in two stages (a and b). From all blocks sections were cut from both of the outer faces (1 and 2). A third, optional, section was cut from the centre of the block after it had been radially split in half (3). 103

FIGURE 21 - Preparation of Microscope Sections for Direct Observations

Sections cut from each radial face (la,b; 2a,b) 3rd, optional, pair (3a,b) cut from centre of split block

Half of ground line block fixed in FAA

Each section stained in safranin/picro-aniline blue and permanent mounts prepared

I I 10 40

3, 10 & 40 mm depths from outer tangential face marked on slide

Perspex grid placed in eyeRiece of microscope to divide the field of view (quadrat) into 9 equal micro quadrats and each one scored. 10 such fields of view scored from specified depth. 104

The actual areas of the sections to be scored were delimited

by a grid. The grid, made of perspex, was inserted into one of the

eyepieces of the microscope. The grid was constructed such that it

divided the field of view of the section into nine equal squares of known

area which were then treated individually (Figure 21).

At the outset it seemed desirable to treat the two wood

species in exactly the same way. However, this became impossible as the

technique developed due to the anatomical differences between the two

species. The rationale was to take a section of uniform thickness which

would represent one complete cell with its contents fixed, but undis-

turbed. This proved relatively simple for the pine since a survey of the anatomical range showed the tracheids to be of a relatively uniform diameter in the tangential plane. The mean tangential diameter of the rrndreids latewoodAwas 27.9 ± 7.6 um and that of the earlywood 32.8 ± 3.1 um, the overall mean diameter being 30.6 ± 7.0 um. Thus, radial sections of pine were cut at 30 um thickness. The major anatomical difference between the softwood and the hardwood was the presence of vessels in the birch. The diameter range of the vessels was 50 — 70 um, whilst the mean tangential diameter of the fibres was 19.39 ± 1.7 um. Ideally, two section thicknesses should have been cut and the thicker, 60 um sections, used to score for vessel characters, and the thinner, 20 um sections, used for fibre properties. However, this became far too time consuming and it was decided that the more important information as regards degradation would be provided by the more numerous, structural fibre elements.

Consequently, the birch was sectioned at 20 um.

3.1.2 STAINING

The sections were stained and mounted using the following regime which was based on Cartwright's technique (Cartwright, 1929) with minor modifications. 105

Having been rehydrated through an alcohol series the sections

were stained in one per cent aqueous safranin for one minute. Excess

stain was then washed out with distilled water before staining in picro-

aniline blue. Picro—aniline blue was made up by mixing four parts of

saturated picric acid with one part of 1% aqueous aniline blue. This

mixture improved immeasurably when left to stand for as long as possible,

two weeks to a month being the preferred minimum. Batches were made up

periodically so that the stain was generally six months old before being

used. A drop of the stain was then placed on the section which was

warmed in a flame just to the point of simmering and held there for a

few seconds before being thoroughly washed- with distilled water. The

sections were then differentiated through 70% and absolute alcohol be-

fore being left in clove oil for at least ten minutes to clear. Finally

the clove oil was washed out with xylene and sections infused with xylene

for two minutes. Each section was then mounted in canada balsam and a

warm cover glass placed over it. The slides were then allowed to dry

completely on a warming tray at 30°C before being 'pressed' and cured in

an oven at 60°C for two days. The pressing was to achieve the uniformly

thin layer of canada balsam along the length of the slide required for

critical high—magnification observations and for photomicrography. This

was achieved by placing a piece of filter paper over the slide and a

thin veneer of wood over this. The whole arrangement was held together

by a bulldog clip which exerted the correct amount of pressure evenly over the slide. After curing, the slides were cleaned of any excess canada balsam and labelled. They were then ready for observation.

The use of safranin/picro—aniline blue was favoured over the many other combinations of stains available. Both stains were applied separately and thus an optimum time for each stain could be determined for the particular material. This is often a serious disadvantage with. 106

double stains such as safranin/fast green where a compromise has to be

reached. Picro-aniline blue will also stain bacteria which fast green

and its allies do not. However, if bacteria were being looked for

specifically a stain such as Gram's stain or carbol fuschin was pre-

ferred. Using safranin and picro-aniline blue, good differentiation was

achieved between the red lignified tissue and the dark blue of the

fungal mycelium, in all cases. The stain did not fade with time as was

found with stains such as Pianese IIIb.

Ultimately the choice of stain must be left to the individual

user since histological staining of plant tissues is a skill which must

be acquired and the advantages of one stain may outweigh those of the

others in a particular circumstance. Various workers have preferred

safranin and fast green (Gram and Jorgensen, 1953; Sorkhoh, 1976) or

safranin and picro-aniline blue (Wilcox, 1964, 1968).

3.1.3 MICROSCOPY AND SCORING TECHNIQUE

For each section random fields or quadrats from specific areas were scored for various characters. The depths initially used were chosen to correspond to 3, 10, and 40mm. from the exposed tangential longitudinal face. These were the depths at which isolations were made from the companion samples (see Section 2.1.2.) and thus provided a good basis for the comparison of the two techniques.

The perspex grid (Figure 21) which was inserted into the eyepiece of the microscope, divided the field of view (quadrat) into nine equal squares. Each of these small squares or microquadrats were then scored for a series of characters so that for each field of view nine microquadrats were scored. A nominal magnification of 500 (x40 objective lens and x12.5 eyepiece lens) was used for Scots pine. This magnification gave suitable detail of tracheid colonization and wall attack. However, this magnification gave insufficient detail of the smaller diameter 107

fibres so that a magnification of approximately 787.5 was used (x63

objective and x12.5 eyepiece lens) for the birch sections.

The characters to be used as a basis of this objective analysis

were derived empirically and divided into four groups (see Figures 22 &

23) .

Firstly, the dominant tissue type present in each microquadrat

was determined and designated by a number. The dominant tissue was that

cell type which occupied greater than 50% of the area of the microquadrat.

Any situation where this could not be assigned with certainty was des-

ignated as being 'outside the classification' and given the character 9

in Group 1. These microquadrats, with a 9 in the first column, were

then ignored in the final analysis, being presented only as a percentage of the total number scored. Clearly if this number was significantly

large it would cast doubt upon the validity of the scoring technique

adopted.

Secondly, the type of hypha, if any, within each microquadrat was characterised. Although several features were considered such as hyphal diameter, orientation within the cell and physical appearance,

the final criterion adopted was that of pigmentation. Thus, the hyphae were scored as either hyaline or pigmented, or both if the area contained examples from each class.

The types of attack were graded from zero (no attack) through

to nine (Basidiomycete wall erosion). This scale (0-9) represented increasing levels of attack. For any one microquadrat a maximum of three

types of decay were chosen from these nine possibilities. If more than

three types of attack were manifest in any single microquadrat, the three most important types, that is the three highest numbers were recorded. Occasions when this occurred were very few so the employment of this constrainst led to no real loss of information. 108

FIGURE 22: DIRECT OBSERVATIONAL CHARACTERS

- PINE -

GROUP 1: TISSUE TYPE

= Tracheids 2 = Ray Parenchyma 3 = Ray Tracheids 4 = Horizontal Resin Canals 5 = Vertical Resin Canals 9 = Outside Classification

GROUP 2: COLONIZATION o = Uncolonized = Hyaline Hyphae 2 = Pigmented Hyphae 3 = Both Hyaline & Pigmented Types

GROUP 3: DECAY

o = No Attack = Pit Penetration 2 = Wall Penetration 3 = Loss of Birefringence 4 = Bacterial Lumen Erosion 5 = Soft Rot Cavities 6 = Basidiomycete Attack (any form) 7 = Bore Holes 8 = Pit Enlargement 9 = Basidiomycete Wall Erosion

GROUP 4: VERTICAL WALLS

1-2: Early Wood 3-4: Transitional Zone 4+ Late Wood 9 Resin Canal

GROUP 5: HORIZ.ONTAL WALLS

1-5: Ray Cells 9 Tracheid End Wall 109

FIGURE 23: DIRECT OBSERVATION CHARACTERS

- BIRCH -

GROUP 1: TISSUE TYPE

1 = Fibres 2 = Rays 3 = Vessels (including wall) ' 4 = Vessels (lumen only) 5 = Fibres + Parenchyma 9 = Outside Classification

GROUP 2: COLONIZATION

0 = Uncolonized 1 = Hyaline Hyphae 2 = Pigmented Hyphae 3 = Both Hyaline & Pigmented Types

GROUP 3: DECAY

0 = No Attack 1 = Pit Penetration 2 = Wall Penetration 3 = Loss of Birefringence 4 = Soft Rot Attack 5 = Wall Decay Type Unknown 6 = Basidiomycete Attack (any form) 7 = Bore Holes 8 = Pit Enlargement 9 = Basidiomycete Wall Erosion

GROUP 4: VERTICAL WALLS

GROUP 5: HORIZONTAL WALLS

1-5: Ray Cells 9 : Fibre End Wall 110

The first three categories represented the more passive

activities of the fungus; no attack (0), pit penetration (1) and wall

penetration (2). Pit penetration (Plates 5.4 & 5.5) could well be the

simplest method of gaining access to the adjacent cell. Destruction of

pit membranes by the fungus is not implicit in this category since mem-

branes may have already been destroyed by the earlier bacterial colonists.

Wall penetration was restricted to those minute penetration hyphae characteristic of the staining fungi which use them, not as a mechanism of food release, but as a means of rapid colonization (Plates 5.1 - 5.3).

Thus, although the cell wall may be penetrated many times no real decay was evident.

The remaining seven categories all represented varying degrees of actual decay.

Loss of birefringence was the decay category used to indicate attack of ray parenchyma in both species.

Category 4 was Bacterial Lumen Erosion. However, for the sample times chosen no evidence of bacterial attack was manifest in

Scots pine. Thus, when scoring the birch samples, which also showed no positive examples of bacterial wall degradation, this category was replaced with another, termed Wall Attack Type Unknown. This was needed because on several occasions, particularly in the untreated birch, heavy decay of the fibres was evident, but the causal organisms could not be determined. Theoretically it could have been caused by bacteria

(Boutelje & Bravery, 1968), or soft rot or Basidiomycetes.

Category 5 represented soft rot attack. This was relatively clear cut in the pine where the decay was in the form of distinct cavities (Plates 6.1 & 6.2), but was less clear in the birch where the attack was seldom in the form of distinct, although irregular, cavities

(Plate 6.3), but as V- and U-shaped sculptured erosion of the wall 111

PLATE 5

Colonization patterns of stainer type fungi.

All specimens are radial longitudinal sections stained in safranin and picro-aniline blue.

5.1 General habit. Progressive colonization either along the rays or

across tracheids in pine.

5.2 Passage of the fungus across a fibre wall in birch by a fine

penetration hypha, much smaller in diameter than the normal hyphae

(decay category 2).

5.3 Passage of Stainers across several fibres in birch (decay category

5.4 Pine. Penetration between tracheids through a bordered pit on the

radial wall (decay category I).

5.5 Pine. Progressive colonization along a ray via pits in the end wall

(arrowed) (decay category 1).

113

PLATE 6

Soft Rot

All specimens are radial longitudinal sections stained in safranin and

picro-aniline blue.

6.1 Typical soft rot cavities in untreated pine. Pigmented hyphae with

proboscis hypha can be seen lying within regular-shaped cavities

(decay category 5).

6.2 As 6.1, but using polarized light.

6.3 Typical appearance of soft rot cavities in untreated birch. The

cavities are variously sized and very irregularly shaped, often

with a single convoluted, pigmented hyphae lying within them

(decay category 5).

6.4 Typical appearance of soft rot in treated birch as V- and U-shaped

sculpturing of the fibre wall, rather than discrete cavities (decay

category 5). 6.3 115

(Plate 6.4). This form of attack was demonstrated in birch by Corbett

using the light microscope and designated Type 2 soft rot attack

(Corbett, 1963, 1965). Recent electron microscope studies have confirmed

that the causal agents are soft rot fungi (Crossley, 1979).

The four final categories represented different forms of

Basidiomycete attack. Class 6 was for any form of Basidiomycete attack,

class 7 for bore holes through the cell wall (Plates 7.2 — 7.4), and

class 8 for bore holes formed via pit enlargement (Plates 7.1 & 8.1).

The final category was for Basidiomycete lumen erosion (Plates 8.2 &

8.3).

Finally, the density of each microquadrat was determined by

counting the number of cell walls within the area of the microquadrat

in both a vertical and horizontal direction. The number of vertical

walls defined whether the field was a mixture of earlywood and latewood

or primarily of one type. The presence of horizontal walls indicated whether the field included a fibre or tracheid end wall (9) or whether ray cells impinged on the field (1 — 5).

Each microquadrat was, therefore, fully described by a seven digit number.

For example in Pinus sylvestris the set,

1 1 6 1 8 ~ 9 5 9 indicated that the microquadrat was composed principally of tracheids (1) which contained hyaline hyphae (1) and showed Basidiomycete attack (6) in the form of bore holes via pit enlargement (8) and wall erosion (9).

Five vertical walls (5) ran through the microquadrat field indicating an area of predominantly latewood. The last figure (9) indicated that the field contained a tracheid end wall. Had this last value been one and not nine this would have indicated a single cross—wall which was not a 116

PLATE 7

Basidiomycete attack in untreated birch

All specimens are radial longitudinal sections stained with safranin and picro-aniline blue.

7.1 Cross-field area showing a large number of bore holes caused through

pit enlargement (decay category 8). Some thinning of the fibre wall

is also apparent.

7.2 Shows a basidiomycete hyphae with clamp connection (c) passing

between fibres through a bore hole across the tangential wall (b.h.)

and via pit enlargement (p.e.) on the radial wall (decay categories

7 and 8 respectively).

7.3' and 7.4 Bore holes through the tangential fibre walls caused by

basidiomycete hyphae showing clamp connections (c) (decay category

7) . 7.1 12

spm 118

PLATE 8

Basidiomycete attack

All specimens are radial longitudinal sections stained with safranin and

picro-aniline blue.

8.1 Typical bore holes in untreated pine caused by enlargement of the

bordered pits on the radial walls of the tracheids (decay category

8) .

8.2 Erosion of tracheid walls caused by Basidiomycetes. The area of

greatest wall thinning is labelled e (decay category 9).

8.3 Basidiomycete wall erosion in birch. A basidiomycete hypha with

clamp connection (c) can be seen lying in its erosion channel (e). 16 pm

~~^ 120

tracheid end-wall. In this case, although the microquadrat was composed

principally of tracheids it abutted an area of ray tissue which just

fell within the area of the microquadrat.

A series of nine such groups of figures precisely defined that

particular field of view or quadrat.

For each section from each depth (that is, 3, 10 and 40mm. from

the exposed face) ten random fields were scored in this manner so that a sample area of 90 microquadrats described one section at one depth.

Initially three sections were cut from each stake (see Section 3.1.1.) and 90 microquadrats were scored in the same way from each of these sections. Thus, at the depth in question, one stake was described by

270 microquadrats. At each sampling time three replicate stakes were used for direct analysis. Consequently, for untreated Scots pine, the information was derived from 810 microquadrats and described the microbiological status of the wood at each time interval.

This analytical procedure was, at this stage, very time consuming. Tests on the collected pine data showed that the least useful replication was that of replicate sections from one stake. Thus, the number of sections cut from the treated pine and the birch samples for subsequent analysis was reduced from three to two without any significant loss of information. This also illustrates the necessity of between- stake replication, that is sampling as many stakes as possible, rather than within-stake replication, that is the number of sections analysed.

Gee Grieg-Smit1, 1964 for full discussion of this hierarchy of replication).

This reduction in the number of sections prepared and analysed from each stake (Figure 21) streamlined the process considerably. Thus=for all the birch samples and for the treated pine samples each stake was described by 540 microquadrats or sets of information.

The advantages of this form of data recording were principally the ease of collection and its flexibility. Data was recorded directly 121

from the microscope slides using a typewriter. This data was then

transferred to computer data cards for analysis.

3.1.4 COMPUTER ANALYSIS

All the information pertaining to one quadrat (a group of nine microquadrats), including a code which identified the position of the

quadrat and its history (sampling time, stake number, section number and depth) was recorded on a single computer card. Replicate information was amassed and analysed collectively and then blocks of information compared.

A computer program (Program ROTTEN) was developed with

Mr. E.F. Baines (Pers.Comm.) to match the flexibility of data collection with versatility of analysis. The program was designed basically to collate large amounts of information, sort and analyse it in a specified way and print out the results. Using various subroutines the analysis was both selective and detailed. Thus, general colonization and decay information could be provided or specific information, such as performance of particular tissue types, the incidence of different types of fungi or modes of attack could be produced.

In this way large amounts of data were processed allowing an analysis of the patterns of colonization and decay in a truly objective manner with any subjective influence minimised as far as possible.

A listing of Program ROTTEN together with a sample set of data and sample output are included as Appendix 3. 122

3.2 RESULTS

Results are presented only for the imm. depth in order to

demonstrate what information may be obtained and how valuable the tech-

nique may prove to be. Attempts can then be made to compare the type

and detail of information provided by this technique with that provided

by the isolation regime and analyses described earlier.

Preliminary investigations were carried out with a small

number of sections covering all sample times in order to observe 1) if

any trends were being established, 2) to obtain a general impression of

the changes that were occurring, and 3) how quickly these changes were

happening. On the basis of this survey together with the results of the

isolation work the three, twelve and eighteen month samples were chosen

to give a complete analysis of each wood species.

3.2.1 PINE

Figures 24 & 25 show the tissue distribution in the scored

microquadrats. These are presented for each sample time, 3, 12 and 18

months, in a similar form; the height of each bar of the histogram being

proportional to the percentage cover of that particular cell type. Thus,

the first histogram in Figure 24 represents the mean tissue distribution

in the microquadrats from untreated stakes sampled at three months.

This revealed that 80% of the total area scored was composed of tracheids,

12% was ray parenchyma and 3% ray tracheid, 2% horizontal and 1% vertical

resin canals whilst 2% lay outside the classification. The second and

third histograms show the same data for 12 and 18 month samples respecti-

vely. Figure 25 shows the equivalent data for treated material.

Figure 26 — 33 represent the more important data extracted from

the complete analysis. The complete analysis, in the form of a computer

printout contains a large bank of potentially valuable information, the most important of which has been transferred to graphs for discussion

(Figs. 26 —.33). Each point on the graph is the mean representation of

123

FIGURE 24 - Untreated Pine: Tissue distribution in scored microquadrats 100-

% RPA) AREA ( AGE IVE PERCENT RELAT •

96 363 591 TIME (DAYS)

Tracheids Ray Parenchyma Ray Tracheids an PP} FIGURE 25 - Treated Pine: Tissue distribution .1s: Horizontal Resin Canals in scored microquadrats Vertical Resin Canals 100- Outside Classification

90-

80-

70-

60-

w 50-

40-

30- da 20-

96 363 591 TT)XT, /TAN/C.\ 124

810 observations in the case of untreated pine and 540 in all other

cases (see Section 3.1.1.). The standard errors from these means are

presented as bars on the graphs in the form of 95% confidence limits.

Figs. 26 & 27 show the graphs for colonization and decay of

untreated pine and treated pine, respectively. These are overall

figures for the whole sample with no account taken of tissue type. Thus

they represent the general level of colonization and degradation in the wood as a whole. It is evident from Fig. 26 therefore, that in the un-

treated pine colonization increased in a linear manner to a maximum of

82% after 18 months. Decay progressed slowly over the first year, only

21% of all tissues showing any sign of degradation, and then rapidly increased over the ensuing 6 months to reach a level of 78%. The values for colonization of treated pine (Fig. 27) showed a close correlation to the untreated material over the first year, in reaching 65% (63% in untreated pine), but they then dropped to a value of 45% after 18 months.

Little degradation, however, was evident, only 7% of all tissue showing signs of any degradation after eighteen months.

Figs. 28 & 29 give a breakdown of where this colonization and decay occurred in terms of the two main tissue types, namely the axial tracheids and ray parenchyma. From Fig. 28 it can be seen that in untreated pine, although the levels of colonization and decay were approximately the same in both tissues at 18 months (means of 79% and 71% respectively for colonization and decay) the routes were slightly different. Colonization progressed very rapidly in the ray parenchyma, reaching

40% by 3 months, peaking at 83% after a year and then dropping slightly to 75% at 18 months. Colonization in the tracheids, however,-was somewhat slower and more regular than in the rays, showing only 8% after three months exposure, but reaching a maximum at 18 months of 84%.

Neither tracheids nor rays showed much decay after a year, although what 125

FIGURE 26 - Untreated Pine: Total colonization & decay

100

90-

T 80-

% RPA) 771 70-

AREA ( 60-

AGE , / 50- i / Colonization , 40- / Decay

IVE PERCENT 30- AT

REL 20- T 10 1 T - ~ 1 0 96 363 591 TIME (DAYS)

FIGURE 27 - Treated Pine: Total colonization & decay % REA (

Colonization AGE A IVE PERCENT RELAT

Degradation , 1 -t - 96 363 591 1'TMT (nAvel 126

FIGURE 28 - Untreated Pine: Colonization & decay in tracheids & ray parenchyma

100-

90- I 80- RPA)

% • irr 70- Ray Parenchyma T ' i'1

AREA ( 60- Colonization '1 , ,Il 50- i / / Ray Parenchyma ~' / i' 40- •f ~' / %/ Decay PERCENTAGE

IVE 30- ,•' Tracheid ' Colonization ,/ /

RELAT 20- i/ Ti / T. Tracheid I ' . 10- Decay__ /

T 96 363 591 TIME (DAYS)

FIGURE 29 - Treated Pine: Colonization & degradation in tracheids & ray parenchyma

100

90-

T Ray Parenchyma

RPA) 80- ,_Colonization % 70 i

60- T •i•. i AGE AREA ( 50- Tracheid r Colonization .0' 40- 1 1

IVE PERCENT 30- i,.,.ð .,.,. 1 AT

REL 20 i Ray Parenchyma, ' Decay ~. I 1 Tracheid 10- .i / • TDegradation T 1 - - 7..-?!.... 1 ..-.1i . 96 363 591 TTMR (nAVCI 127

there was seemed to be chiefly in the tracheids (10% and 1% respectively).

Between 12 and 18 months, however, decay proceeded very rapidly in both

tissues with 79% of tracheids and 63% of ray parenchyma cells showing

signs of decay at 18 months.

Comparable figures for the treated pine (Fig. 29) revealed

quite a different picture. Ray colonization seemed very rapid, reaching

57% at 3 months, although the standard deviation was quite large, rising

to 78% at 12 months. Tracheid colonization increased to 22% at 3 months,

again with a relatively large standard deviation, rising to 60% after

one year. In the following 6 months, however, the levels of colonization dropped in both tissues, to 67% in rays and 43% in tracheids. Figures for decay remained low throughout, reaching only 5% in tracheids and

26% in rays after 18 months.

Figs. 30 & 31 show how much of the decay was attributable to either soft rot fungi or to Basidiomycetes. Fig. 30 shows levels of soft rot and basidiomycete attack of any sort in all tissues, whereas

Fig. 31 shows the same figures for tracheids only. Both of these apply to the untreated pine since the treated pine revealed no decay attributable to either Soft Rots or to Basidiomycetes even after eighteen months exposure.

Fig. 30 shows very little 'major' decay at 12 months in all tissues. In fact, the only evidence is the presence of soft rot cavities in 1.5% of the cells, no basidiomycete decay being observed. However, in the ensuing 6 months 27% of the cells showed soft rot cavities and

59% had been attacked by Basidiomycetes. From Fig. 31 it was evident that the major part of this decay was of tracheid tissue.

The type of colonization can be further categorised to show- the relative importance of different types of hyphae in the colonization process. Figs. 32 & 33 show the type of information that can be gained from this consideration. The two figures show the relative occurrence 128

FIGURE 30 — Untreated Pine: Soft rot & basidiomycete attack in all tissues

100

90 fcZ 80

60 ci H w 50 V LY.. ā 40 Basidiomycete H 30 Attack 20 ,%I /Soft Rot 10 Cavities

0 96 363 591 TIME (DAYS)

FIGURE 31 — Untreated Pine: Soft rot & basidiomycete attack in tracheids

100

PA)

R 80 Z 70

50 Basidiomycete 40 Attack

30 ATIVE PERCENTAGE AREA (

REL 20 'Soft Rot

10 ,' Cavities

96 363 591 TT1tE^ ITAVON 129

FIGURE 32 - Untreated Pine: Types of colonizing hyphae in all tissues

100-

T 80- % RPA) 70- e T ee 60- Total '1.

AGE AREA ( Colonization / 50 i /Hyaline 40 i /• Hyphae /

IVE PERCENT 30- V.

RELAT 20- T Pigmented & 1 ' I Hyaline Hyphae • 10- j~~ -fr- Pigmented Hyphae 0 96 363 591 TIME (DAYS)

FIGURE 33 - Treated Pine: Types of colonizing hyphae in all tissues

100

80 % RPA) 70 T 60 /' S.

AGE AREA ( Total /.. • 50 Colonization // I ENT /

40 / ' ~'—1 • 1 ,./ i• - 1 Hyaline IVE PERC 30 fi ' AT

REL 20 ll x / I Pigmented Hyphae 10 <47

_ _. 1 _ ;Hyaline & Pigmented • Hyphae 96 363 591 TTM. (TIAVS) 130

of hyaline and pigmented hyphae individually and combined in both un-

treated and treated pine. Fig. 32 shows the untreated case where there

was very little difference in numbers between the two types of hyphae

during the first 12 months. However, after 12 months the presence of

pigmented hyphae dropped from 24% to just 3% by 18 months. In contrast

the hyaline hyphae increased from 29% to 60% over the same period.

Occurrences of pigmented and hyaline hyphae together rose steadily with

time to 18% at 18 months.

In the treated pine (Fig. 33) there seemed to be very few

fungal hyphae with pigmented walls. Even after 12 months only 16% of

all cells showed their presence, rapidly dropping to 6% at 18 months.

Levels of colonization by hyaline hyphae were generally higher, rising

to 41% at 12 months and then dropping marginally to 37% at 18 months.

Cells containing hyaline and pigmented hyphae remained sparse, never

exceeding 10% throughout the study.

3.2.2 BIRCH

Fig. 34 shows the mean tissue distribution in scored micro— quadrats in untreated stakes sampled at 3, 12 and 18 months-, whilst Fig.

35 shows the equivalent data for the treated stakes. Thus, at three months, for example, as shown in the first histogram in Fig. 34, 63% of the area scored consisted of fibres (total fibres). Of this total fibre area, 52% were fibres only, whilst 13% were fibres plus- parenchyma.

15% of the area consisted of ray tissue and 4% lay outside the classification. The remaining 18% was composed of vessel material of which.

7% was lumen only and 11% included some part of the vessel wall.

Figs. 36 — 49 represent the main data provided by the analysis of the untreated and treated birch samples at 3, 12 and 18 months.

As previously described for the pine samples (Section 3.2.1.) each point on the graph is the mean representation of 540 individual observations. The standard errors from these means are presented as bars 131

FIGURE 34 - Untreated Birch: Tissue distribution in scored microquadrats 100

-< ~ N '-' < ~ t:l 60 C,!) ~ z rz:3 50 U ~ rz:3 ~ 40 ~ . >..-t ~ 30 < ...1 ~ 20

o 96 363 591 TIHE (DAYS)

Fibres Fibres + Parenchyma Total Fibres Rays FIGURE 35 - Treated Birch: Tissue distribution in scored microquadrats Vessel (inc. lvall) Vessel (excl. Wall) 100 Outside Classification

o ~96 363 . 591 TTMli' (n&vc:) 132

on the graph to form 95% confidence limits.

Figs. 36 & 37 show the graphs for colonization and decay of

untreated and treated birch respectively. They represent the levels in

the wood as a whole with no account taken of constituent tissues. Thus

colonization increased fairly rapidly in the untreated to 29% after 3

months exposure and further colonization increased with time reaching

60% after 18 months.

Colonization of treated birch increased in a linear fashion

to a final level of 56% after 18 months.

Total decay in the untreated birch progressed steadily until

66% of all tissues showed some signs of attack after 18 months. At 3

months less than 1% of the treated material showed any form of decay.

However, after this initial lag period the degree of attack increased in

a way comparable to that of the untreated, reaching 54% after 18 months.

As shown in Figs. 34 & 35 birch wood is composed of four main cell types; fibres, rays, axial parenchyma and vessels. Figs. 38 - 49 show the effects of colonizing fungi on these individual tissues.

Parenchyma was not scored directly because it constituted less than 5% of the total bulk and this was in scattered bands so that it never formed the dominant tissue type in any microquadrat. However, its presence was recorded by the fibre plus parenchyma category, but in this category it was the fibres themselves that were scored.

Figs. 38 & 39 show the rate of colonization and decay for untreated and treated material respectively for both categories (fibres and fibres plus parenchyma). t-tests revealed non-significant differences between the levels of colonization and decay in the two: categories at all sample times in the untreated birch. Consequently the two sets of measurements were averaged and the means presented in Fig. 40 as total fibre colonization and decay. t-tests on the treated material revealed two cases where the values of t were just marginally significant. 133

FIGURE 36 - Untreated Birch: Total colonization & decay

100-

90-

80- % RPA) 70- 60- 1 , 50- Colonization 40 PERCENTAGE AREA (

IVE 30- r Decay RELAT 20- 1

10 'l 0 96 363 561 TIME (DAYS)

FIGURE 37 - Treated Birch: Total colonization & decay

100

90-

RPA) 80- % 70

60- AGE AREA ( i, 50- i i i i 40- i i IVE PERCENT 30 Colonization i

RELAT 20- , ~Decay 10- /

96 363 591 TT.T /T •~se+\

134

FIGURE 38 - Untreated Birch: Colonization & decay in fibres (F) and fibres + parenchyma (FP)

100

90-

PA) 80- % R 70-

AREA ( 60 Colonization (F) 50-

40- I

30-

RELATIVE PERCENTAGE 20

10-

363 591 TIME (DAYS)

FIGURE 39 - Treated Birch - Colonization & decay in fibres (F) and fibres + parenchyma (FP)

100- 11.

90-

RPA) 80 % 70

60- , / / AGE AREA ( ' i 50- t// i ENT

40- Colonization /• i ! (F) /• - i /Decay (F + P) Decay i / 30 +P) /, (F -/ i. ! %at~on' i RELATIVE PERC 20-

10-

96 363 591 TT.m !n♦[7n\ 135

Comparison of colonization and decay at 12 months revealed values for t

of 0.050 and 0.041 respectively. (Values of t less than 0.05 indicate

significance at the 5% levels.) However, these differences were con-

sidered too small to justify totally separate treatment of the two groups

and the two sets of data were averaged in a similar manner to the un-

treated birch and are presented in Fig. 41 as total fibre colonization

and decay.

Fig. 40 indicates that colonization of total fibres occurred

very rapidly in the untreated birch, reaching 37% by 3 months with fungal

mass increasing steadily until 80% of the fibre area showed signs of

colonization at 18 months. Decay similarly increased to 91% at 18

months.

Fig. 41 presents a remarkably similar picture for treated

material. Colonization increased gradually to a level of 72% and decay,

after an initial lag period of 3 months, to 76% of the total fibre area

at 18 months.

Figs. 40 & 41 both show decay in general terms, that is, any

form of attack. This is divided into the major forms of wall attack in

Figs. 42 & 43, that is, decay caused by Basidiomycetes, by Soft Rots and

that which could not be assigned to either with any degree of certainty

due to its advanced nature (termed "type unknown"). Thus, the results from untreated material showed that soft rot was not very important in

terms of volume of decay, less than 20% of the total sample area showing any sign of soft rot attack at any time. The major form of attack was that caused by Basidiomycetes. Although appearing after the first signs of soft rot, basidiomycete attack rapidly increased to 66% by 18 months.

Less than 15% of the attack could not be assigned to either category.

Fig. 43, the equivalent for treated birch shows, in complete contrast to the untreated case, soft rot to be of extreme importance with all the observable decay attributable to soft rot fungi. No evidence of basidiomycete attack was manifest. 136

FIGURE 40 — Untreated Birch: Colonization & decay in total fibres

100-

90- T

cr+ 80- sse -4 70- t 60- cw7 Colonization H 50- / / ā 40- 1 w / Decay 30- / / 20- / /

10-

96 363 591 TIME (DAYS)

FIGURE 41 — Treated Birch: Colonization & decay in total fibres

100-

90-

80 % RPA) 70

60-

50- Colonization 40- / / / IVE PERCENTAGE AREA ( 30- //Decay

AT / /

REL 20

10-

916 363 591 TTMR (11AVG) 137

FIGURE 42 - Untreated Birch: Major types of wall decay in total fibres

100-

90-

A) P 80- 7 R 70- EA (

AR 60 AGE

ENT 50-

40- Basidiomycete IVE PERC 30- Attack

RELAT 20- 11 Soft Rot Decay -• Type Unknown •--- ___ 10- imm' 'I ,~•-- • .+

0 96 363 591 TIME (DAYS)

FIGURE 43 - Treated Birch: Major types of wall decay in total fibres

100-

90-

80- I I 70- , I.../ /. W 60- ,'Soft Rot d . •. Decay w 50- U P4 / ā 40- ~' H 30- a 20- i

10- •' -- --- 0 9'6 363 591 • TTM !TANG\ 138

Figs. 44 & 45 show the rate of colonization and decay in the

ray parenchyma of untreated and treated birch respectively. Colonization

was erratic and remained low, in the untreated material, showing 26% of

the area colonized at 12 months, but only 7% at 18 months. Decay, as

represented by loss of birefringence, steadily increased to 30% at 18

months. In the treated material colonization remained at low levels,

only 11% of the area showing signs of colonization at 18 months and decay

was manifest only at 18 months when it had reached 26%.

Vessel colonization is shown in Figs. 46 & 47. No attack was

evident in any samples. The colonization data is presented as scored,

that is, separated into vessel areas including wall and those excluding wall so that only the lumen was present in the scored microquadrat. In

both treatments, not surprisingly, the lumen only category showed lower levels of colonization than in cases where walls impinged on the scoring area. In the untreated material colonization reached a peak at 12 months with 18 month samples showing lower levels of colonization. In contrast colonization of the treated birch continued to rise with time.

Figs. 48 & 49 present the degree of colonization in terms of the percentage of colonizing fungi possessing pigmented and non—pigmented walls. Fig. 48 shows this information for the untreated birch where it is evident that the major component of the colonizing hyphae lacked any pigmentation. Hyphae possessing pigmented walls, whether in isolation or mixed with hyaline hyphae, represented only 27%, 18% and

28% of the total colonization respectively at 3, 12 and 18 -months. In contrast this same category accounted for 15%, 40% and 51% of the total colonization in the treated material (Fig. 49). 139

FIGURE 44 — Untreated Birch: Ray colonization & decay

100-

80- % RPA) 70-

50-

40-

1 IVE PERCENTAGE AREA ( 30- Decay I I I

RELAT 20 Colonization 10-

0 96 363 591 TIME (DAYS)

FIGURE 45 - Treated Birch: Ray colonization & decay

100-

80- Z RPA) ( 70-

AREA 60- AGE 50 ENT

IVE PERC 30- •Decay i RELAT 20-

10- i~ ,-Colonization ' i 1 0 96 363 591 TrMV (nevc\ 140

FIGURE 46 - Untreated Birch: Vessel colonization

100-

90-

80- RPA) % 70-

60- AGE AREA ( 50- I 40 Vessel (inc. Wall)

IVE PERCENT 30- AT 1 REL 20- Vessel (exc. Wall) 10 T~

96 363 591 TIME (DAYS)

FIGURE 47 — Treated Birch: Vessel colonization

100

w 60 z 50 Ū Vessel (inc. Wall) R9 40

Ē, 30 Vessel (exc. Wall) 20 10 --- 96 363 591 TTMF (nev¢1 141

FIGURE 48 - Untreated Birch: Types of colonizing hyphae in all tissues 100

90-

80- % RPA) 70- 60- T___ -.Total1 Colonization 50- . ~- .._._...... T_.. Hyaline Hyphae 40- ./•1 I T /. 30- I / ATIVE PERCENTAGE AREA (

REL 20 / - i. // THyaline & Pigmented 10- '_Hyphae j T 1 'Pigmented Hyphae i 0 96 363 591 TIME (DAYS)

FIGURE 49 - Treated Birch: Types of colonizing hyphae in all tissues

100-

90-

80 % RPA) 70

60- T Total Colonization 50- ~1 40- T,

IVE PERCENTAGE AREA ( 30- .'~

AT _ :Hyaline Hyphae . REL 20- ∎ i T~ ~.i • 1 . Pigmented Hyphae 10- ~,1 •~ T I Hyaline & Pigmented -.---- •"Hyphae

0 96 363 591 TTM' fnevcl 142

3.3 DISCUSSION

The scoring regime produced figures representing relative areas

of colonization and decay. This is quite distinct from numbers of cells

colonized and degraded. It must, therefore, be borne in mind that a

figure of 80% tracheid colonization represented 80% of the total area of

tracheids scored being colonized by fungal hyphae and not 80 out of every

100 tracheids being colonized. Because of the nature of the wood, being

predominantly long, thin cells, and the nature of the scoring method, any

one cell could be scored more than once. However, each value represented

a different area of that cell and thus presented valid information. This meant that sample areas scored were quite small (microquadrats),.perhaps analagous to point-sampling in higher plant ecology. Multiple samplings of this kind, however, lead to a much more realistic appraisal, since the presence of a single hypha or a small area of attack did not get magnified and lead to that cell being classified as colonized and/or decayed. Due to multiple sampling any one cell would need to be colonized and/or show signs of attack along its whole length before it was classed as truly colonized or decayed. For example, a single soft rot cavity in one tracheid would be picked up by perhaps one out of three microquadrats whereas if a long chain of cavities were present these would show in all three microquadrats. The two results would be quite different. In both cases the tracheid is degraded, but by multiple small samples (microquadrats) the former case would be assigned a value of 33% and the latter, with more evidence of wall breakdown, a value of 100%. This demonstrates the usefulness of Relative Percentage Area, hereafter referred to as RPA, rather than an assessment based on presence or absence.

In the ensuing discussion all figures presented represent relative percentage area (RPA).

For the main analysis the values of each character in the 10 quadrats (90 microquadrats) were averaged. This was then counted as one 143

observation, the 90 microquadrats being sub-samples relaying the full

information for that particular section. Either two or three sections

per replicate stake (3 stakes) were analysed at each time interval giving

six or nine 'observations'. These observations, when pooled for the

final analysis, then represented the sum of either 540 or 810 micro-

quadrats scored. These were then represented by one point on the graph.

Tests of significance (two-tailed t-test) were performed where

required (Sokal & Rohlf, 1969). The Null Hypothesis was that the two

means for comparison were assumed to be equal unless the Probability of

t was less than 0.05. This established with 95% confidence limits that

the two values were sufficiently different for it to be assumed that they

were derived from different populations.

3.3.1. PINE

The histograms of tissue distribution in the scored micro-

quadrats (Figs. 24 & 25) showed a uniform distribution of tissue in all

samples regardless of time and treatment. No significant difference

between them could be demonstrated. This indicated that all samples

were derived from a uniform population so that any differences in per-

formance could be attributed to the treatments and not to the starting material.

In the untreated pine (Fig. 26) total colonization gradually increased with time in a linear fashion. Thus fungal mass increased as the fungi steadily colonized more of the tissue, 82% of the tissue area being colonized by 18 months. Whether this would eventually increase to

1002 and how long it might take are conjectural.

The decay followed a typical exponential curve. There seemed to be a lag period of 12 months during which time the decay organisms became established, displacing the earlier, more passive inhabitants (see.

Section 2), and built up sufficient numbers, or inoculum potential, to 144

break down the wall materials to a degree which could be detected by

the technique. Once established and decay initiated, the process entered

a log phase, after which decay proceeded very rapidly between 12 and 18

months, until 88% RPA of all tissues showed some sign of decay.

Colonization and decay reached approximately the same level by

18 months. This would be expected from the evidence that until hyphae

are present there can be no decay. Since it took time for decay to be

manifest, equal RPAs for colonization and decay probably meant that the

colonizing fungi were wood-rotters. This would infer that every cell

colonized was decayed which may be an over-simplification since some cells

may be colonized and not attacked; both colonized and decayed; and others

may be attacked but show no signs of colonization due to hyphal death.

This would also result in equal RPAs in the same way as would every cell

colonized being attacked.

Treated pine (Fig. 27) revealed quite a different pattern as

far as total colonization and decay were concerned. For the first year,

however, the colonization data presented a very similar pattern to that

in the untreated pine. In fact, it would appear that colonization had

been stimulated by the presence of CCA since 27% of the tissue area showed

evidence of colonization at 3 months in the treated sample-as compared to

13% in the untreated. This could represent copper-tolerant organisms

growing quickly because of the lack of competition with non-copper-tol-

erant organisms. However, this was not a significant difference (t =

0.710), thus giving no evidence to justify stimulation nor an increased

level of colonization. Between 12 and 18 months there was a significant

drop in the level of colonization from 65% to 45% indicating that fungal mass had disappeared (t = 0.028). Presumably any soluble food sources upon which the inhabitants had been living had been exhausted and the fungi died through lack of nutrients since, there being no decay, they were unable to Utilize the substance of the wood itself. This period 145

also coincided with the onset of rapid decay in the untreated material.

However, this could explain the plateau in the graph, but not the drop,

since, even if dead, one would still expect the fungi to be present and

thus scored. The drop represented a loss in fungal material. Three

explanations of this phenomenon are acceptable. Fungi could be para-

sitized by other fungi which would reduce fungal mass. This has been

observed in this material (see Plates 9.1 - 9.3). Alternatively, hyphae,

once dead, may have autolysed and consequently be unrecognizable as

structured hyphae, merely as debris. More likely, active hyphal front

has grown further into the wood, scavenging for food, autolysing the older parts and no re-infection has taken place.

A sequence of this nature has been observed in many of the complete radial longitudinal sections:

I I Outside 1 I I I Inside I 1 (open to 1 1 1 (resin Environ- I I covered) ment) no . pigmented hyaline no hyphae hyphae hyphae bacteria colonization

Direction of colonization

Fig. 50 Approximate distribution of organisms across treated stakes

showing radial penetration and no re-infection from the

environment.

In total contrast to the untreated material, the treated pine showed very little degradation. Even after 18 months exposure only 7% of the tissue area showed any signs of attack. In fact, all this was

'colonization degradation' rather than actual decay, that is, passive pit penetration (see Plates 5.4 & 5.5) and wall penetration (see Plate 5.2 &

5.3). This has affected the wall, however little, and, therefore, needs 146

PLATE 9

A possible case of fungal parasitism

9.1 - 9.3 Radial longitudinal section from an untreated pine stake

stained with safranin and picro-aniline blue. The photographs

are of a ray area and show one fungal hypha growing inside another.

This could either be parasitism, or saprophytism if the fungus was

already dead. It is unlikely to be re-growth through an old hypha

since the two hyphae show different features. The outer hypha

shows some natural pigmentation characteristic of the stainer-

type fungi whilst the inner one is a hyaline type. The inner

hypha also shows a very short side branch which could be a clamp

connection (c) showing it to be a basidiomycete. The only bas-

idiomycete isolated at 6 month from untreated pine was Sistotrema

brinlananii which showed no decay reaction on sawdust. Thus it may

be gaining its nutrition parasitically, or saprophytically, from

the earlier staining fungi. The inner hypha also shows a charac-

teristic constriction as it passes through a septum (s) of the

outer hypha.

As discussed in Section 2, S. brinkmanii has been shown to be

a parasite of other fungi in wood and as yet has not been shown to

be a wood-destroyer. Therefore, it is possible that it is merely

living on other fungi in the wood and not the wood itself. 9.1

•*"

• • do.

9.2 , 5PM,

9.3 sum 148

to be considered as attack, but since it does not affect the strength of

the wood (Savory & Pinion, 1958) it is of little importance.

Fig. 28 showed colonization and decay of untreated pine in

terms of its two main cell types; ray parenchyma and axial tracheids.

The two tissue types acted quite differently in terms of colonization

rates, but remarkably similarly in terms of decay. Colonization of the

rays was very rapid, reaching 40% by 3 months and increasing to 83% by

12 months. It then dropped slightly to a mean value of 75%. However,

this drop was not statistically significant (t = 0.139). From the iso-

lations (Section 2.2.1.) it was revealed that most of the organisms

present between 12 and 18 months were Basidiomycetes which have been

shown to be capable of autolysis of the older hyphae and the controlled

translocation of the breakdown products to be re-utilized at the growing

tips (Jennings, Thornton, Galpin & Coggins, 1974; Levi & Cowling, 1969;

Merrill & Cowling, 1966; Savory, 1964; Weigl & Ziegler, 1961). Since the

hyphae have gained entry via the rays (see below) and then into the

tracheids, the drop in colonization values may represent an export of

material from the rays to the tracheids. From these results it therefore

appears that the area of ray parenchyma colonized remained static or

dropped slightly between 12 and 18 months. Colonization of the tracheids

was significantly lower than the ray parenchyma until 18 months.

The graphs of individual tissue decay (Fig. 28) presented a

similar picture to each other and to that of the tissue as a whole

(Fig. 26). Again there was a long lag period with no decay evident.

Then between 12 and 18 months the level of decay increased markedly.

There seemed to be slightly greater levels of decay in the tracheids than

in the ray parenchyma. This was contrary to expectations since greater

protection should have been afforded to the heavily lignified tracheids

than to the unlignified ray parenchyma cells. Although the differences

are not statistically significant, in relation to the differences in 149

chemical composition between ray parenchyma and tracheids, amore likely

value would have been markedly higher levels of attack in the ray paren-

chyma tissue. However, this may reflect the techniques employed.

Monitoring of tracheid decay was quite sensitive since the various cate-

gories are easily observable in RLS and relatively clear-cut and hence

scored. Therefore, levels as low as 1% Cat 96 days) and 10% Cat 363 days-) can be detected. Loss of birefringence as a criterion for decay is much more insensitive and difficult to score since it relies on a good

polariser/analyser combination and a black background for clear Bire- fringence, which is very difficult to achieve in stained material. It proved much more useful on unstained material in transverse section.

The unlignified ray parenchyma was more easily damaged during sectioning since the material was not embedded, which could have affected the results. Despite these drawbacks loss of birefringence remained the most useful criterion for detecting attack of ray tissue.

Colonization of treated tissues, both tracheid and ray parenchyma (Fig. 29) paralleled that of the untreated tissues, in the early stages. t-tests showed non-significant differences between the levels of colonization in both tissue types up to and including one year.

Therefore, it seemed that the introduction of the preservative made no difference to colonization in the first year. However, as- will Be referred to later (see Section 4) results from the Isolation Techniques

(see Section 2) have shown that, although there was no difference in terms of gross colonization, as indicated by- Direct Observation, there had been severe selection, either in the soil or in the wood, for different groups of fungi, the resulting floral composition being vary different.

In the ensuing six months direct observation revealed a drop in colonization in both tissue types. The drop in ray colonization was not significant (t = 0.143) whilst that in the tracheids was marginally non- significant (t = 0.056). The degradation that was present was confined 150

mostly to the ray parenchyma and, even after 18 months it had reached

a level of only 25%. This value represented a loss of birefringence in

25% of the ray area scored (3% of the total area) and may represent the

initial stages of decay. However, as discussed earlier, this criterion

is somewhat unreliable in the early stages of decay. Tracheid de-

gradation at 18 months was 5% and all of this was attributable to passive pit— and wall—penetration.

There was no evidence of any attack by either soft rot organisms

or Basidiomycetes in any of the treated samples. Therefore, in terms of

structurally debilitating attack, the treated pine stakes, after 18

months in the field, were still totally sound.

Fig. 30 showed the RPAs of all tissues showing soft rot cavities i or basidiomycete attack in the form of wall thinning, bore holes or pit enlargement, in the untreated samples. These represented the 'major'

types of decay, that is, those which affect the structural strength of the wood and can thus eventually lead to failure of the timber. Soft rot organisms colonized the stakes first and the effects of their attack, in the form of cavities, were first evident at 12 months, although below-5%, gradually building up to 27% at 18 months. Breakdown due to Basidiomycetes was not evident until 18 months when it had already built up to 59% of the sample area. These two results agree with the physiology of the organisms and their method of attack. Soft rot attack is characterist- ically a slow process, passing from one cell to the next, breaking down most of one cell before invading the next, as demonstrated in these sections. The number of soft rot organisms, as revealed by isolations, also increased slowly. Conversely, the Basidiomycetes colonized rapidly, appearing almost simultaneously throughout the block with a consequent rapidity of attack.

Fig. 31 shows the same information as Fig. 30, but in terms of tracheid attack, rather than of all tissues. This revealed that most of 151

the soft rot and basidiomycete attack scored was in tracheid tissue.

Ray tracheids acted like the axial tracheids, showing some bas-

idiomycete attack (20%), but the relative area of ray tracheids compared

to axial tracheids was very small. Ray parenchyma showed no attack

directly attributable to Soft Rots or Basidiomycetes; the loss of bire-

fringence could have been caused by either type of fungus.

Additional information was obtained about colonization by

further dividing colonizing hyphae into types. The simple criteria of

whether the hyphae had pigmented or hyaline walls was chosen as a potenti-

ally useful, easily scored character. Fig. 32 showed the relative distri-

bution of these two types Of hyphae both singly and in association in the

untreated samples. Fig. 32 showed that the relative occurrence of

pigmented and hyaline hyphae was the same up to 12 months. Cases of

pigmented and hyaline hyphae occurring together showed a similar increase

up to twelve months, but at lower levels. Between 12 and 18 months the occurrence of fungi with hyaline walls increased markedly at the expense of those with pigmented walls which dropped to less than 5%. Cases of both types together increased slightly, but remained below 20%. This would seem to indicate a disappearance of the staining and soft rot organisms with the increase of hyaline hyphae representing Basidiomycetes and Secondary Moulds. Distinction between the latter two groups could be made on two counts. The first by subjective inference as to their position in the cell and their relationship to the decay pattern around them. Secondly, on many occasions clamp connections could be observed along the length of fungi within wood cells (e.g. Plates 7.2 & 7.3), thus identifying them as Basidiomycetes. This could have been the: basis of an objective criterion for scoring the presence of basidiomycete fungi within the wood. However, clamps were difficult to detect without increasing the magnification, which was time-consuming, and were often widely spaced on 152

the hyphae or simply absent as in the monocaryon stage of the life—

cycle which is very frequent in nature (Rayner and Todd,1979; Gilbertson,

1980). Thus, in terms of a reproducible criterion, presence of clamps

was neither reliable nor rapid enough, whilst remaining a useful sub-

jective character.

Fig. 33 showed similar information in treated wood. The

isolation work has revealed the treated material to be full of stainer/

soft rot type organisms possessing pigmented hyphae and this would seem

to be contradicted by the direct observation data as presented in Fig. 33.

This showed low levels of colonization generally, but particularly of

fungi with pigmented walls, hyaline hyphae being at significantly higher

levels at 12 and 18 months (t = 0.013 and 0.009 respectively). Many of

the hyaline'hyphae scored may represent very young Soft Rots which have not yet laid down any pigment. Cultural studies have shown that many of

the pigmented Soft Rots isolated do not produce wall pigment in the very young, leading hyphae, but only at some distance back, often several mil- limetres. Thus, this rather simple character of pigmented/non—pigmented hyphae in wood needs to be interpreted very carefully since, on the face of it, it would appear to be a useful indicator of the type of organisms present in the untreated, but not in the treated material.

From the preceeding discussion certain trends may be defined:—

I. The colonization rates of treated and untreated pine

were similar for the first year after which time the

rate of colonization of untreated continued to rise

whilst that of treated dropped.

2. The primary route for colonization in both treated

and untreated wood was the ray parenchyma, tracheid

colonization being consistently less frequent. The

colonization of ray parenchyma was very rapid in 153

the early stages up to three months.

3. No decay of any description was found in treated

pine samples.

4. Decay levels in the untreated pine were very low

during the first year, increasing very rapidly

between twelve and eighteen months.

5. Both soft rot cavities and basidiomycete attack

were evident in untreated pine at eighteen

months, but levels of basidiomycete attack were

more than double those of Soft Rots.

6. The presence of CCA had a profound effect on

both colonization, by reducing it, and decay,

by completely inhibiting it.

3.3.2 BIRCH

The histograms of tissue distribution (Figs. 34 & 35) reflected

a similar pattern in all samples indicating that the samples came from a

uniform population and can be compared as such.

Figs. 36 & 37 presented strikingly similar pictures of both.

colonization and decay in untreated and treated birch respectively. This

was confirmed by t-tests which showed there to be no significant differ-

ences between them in any respect. Even the apparently low levels of

decay in treated birch at three months (0.5% RPA) was not significantly

lower than the untreated mean of 10.4 % RPA (t = 0.112). This was due

solely to the large standard deviation of 12.6 found -fGr the decay

value of the untreated. This large standard deviation is a reflection of

the patchy nature of the initial stages of decay where some areas were

sound whilst tissue areas immediately adjacent showed signs of attack.

From Figs. 36 & 37 it can be inferred that the introduction of

CCA into the birch had caused no significant reduction in either the rate 154

of colonization or the initiation of decay and its subsequent develop-

ment.

Total fibres constituted an average 63% of all samples scored,

treated and untreated, and these became heavily colonized relatively

early. By 18 months 80% of the untreated and 72% of the treated fibre

area was colonized (Figs. 40 & 41). Decay also proceeded rapidly in the

fibres, once initiated. As the fibres constitute the main bulk of the

wood, both in terms of volume and structural strength this rapid attack

of the fibres would lead to a very short life for the timber in service,

even when treated.

The fundamental difference between the two treatments was ex-

emplified by Figs. 42 & 43 where the types of fungi causing the observed

decay were segregated. Fig. 42 showed the case in untreated birch where

soft rot was evidently a minor component and it was the basidiomycete fungi which were the more able competitors attacking the wood directly despite the fact that the Soft Rots were already established before any signs of Basidiomycetes were evident. Between 3 and 12 months 47% of the total fibre area showed signs of basidiomycete attack with a further

12% attack of uncertain origin. The basidiomycete attack had increased to 66% by 18 months, with a similar 16% of uncertain origin. Soft rot attack fluctuated between 10-20% df the total area.

The situation in the treated material was in marked contrast to that presented by the untreated wood. Basidiomycetes were neither isolated (Section 2.2.1.) nor was there any decay attributable to them.

Once Basidiomycetes had been eliminated from the succession, by the presence of CCA, the soft rot fungi became dominant. Therefore, after

12 months 50% of the total fibre area showed signs of attack and by 18 months 73% showed soft rot cavities or erosion of the soft rot type.

Thus, if Basidiomycetes were considered the climax of the succession in 155

untreated wood, being the most vigorous competitors, soft rot fungi had

become the climatic or plagio—climax of the succession in treated wood,

CCA being the abiotic component which had suppressed the Basidiomycetes.

This is in agreement with the situation encountered in the isolation

studies reported in Section 2.2.1.

Figs. 44 & 45 indicated that the ray tissue of birch was not

as important as the ray tissue in pine as a primary route for colonization.

Levels of colonization remained low throughout. In the untreated birch

the levels were well below that of the fibres, reaching a peak of 26% at

12 months and dropping to only 7% at 18 months. This drop at 18 months

corresponded to a peak decay of 30%. This decay often represented a

complete loss of structure of the rays so that fungus, even if present,

could have been lost in sectioning as there was no wall to adhere to.

The drop in colonization may alternatively represent an export of fungal material by growth to other tissues of the wood.

In the treated birch (Fig. 45) colonization was extremely low,

the rays showing no signs of colonization at 3 months and only 3% and

11% RPA respectively at 12 and 18 months. Decay was only apparent at

18 months when 26% of the ray area scored showed a loss of birefringence.

Most of the values for colonization and decay had high standard deviations, again reflecting an uneven distribution of both the tissue and consequently colonization and decay within them.

The scoring of vessels presented a problem unique to itself.

The average cross sectional diameter of a vessel was 60 um and that of a fibre 20 um. Therefore, two section thicknesses were required to obtain maximal information, that is, one at 20 pm for the fibres and:rays and a second one of 60 um thickness for the vessels. However, this was impossible due to the limitations of time. Therefore, a section thickness of 20 um was chosen as fibres were considered as more directly important since they constituted the structural element of the wood, that is, its 156

properties as a timber (see Section 3.1.1.). This inevitably meant that

information was going to be lost since only parts of vessels would be

contained within the plane of any section, some of its contents being

lost during sectioning. In fact, so great was the discrepancy in size

that areas were scored which were just the centre of a vessel with no

attached wall and contents were obviously underestimated in these cir-

cumstances. This can be inferred from the results in Figs. 46 & 47

where the 'lumen only' category showed consistently lower levels of colonization than vessels including walls.

The initial levels of colonization of vessels (including walls) of 33% RPA and 17% RPA at 3 months in the untreated and treated respectively are comparable with levels of colonization found in the fibres

(37% RPA and 15% RPA). If vessel colonization is considered to be underestimated for the reasons outlined above, together with the qualitative observations of heavily colonized vessels it can be inferred that the vessels act as a primary route for colonization. This would confirm earlier, qualitative observations of the role of vessels in initial colonization (Wilcox, 1968; Bravery,1972). However, to be quantified, a further set of thicker sections which included the whole contents of the vessels need to be scored in a similar fashion.

The vessel walls in both untreated and treated birch showed no signs of decay even though by eighteen months the tissues surrounding them, particularly the fibres, were heavily colonized and attacked. This freedom from attack in untreated vessels has been earlier reported by

Greaves (1966) using the light microscope and George (1974) using the scanning electron microscope. They reported birch vessels to be colonized, but not attacked, whilst the surrounding fibres were badly decayed.

The final two sets of results presented for birch (Figs. 48 &

49) showed the analysis of colonization in each treatment. Fig. 48 for untreated birch fitted in well with results previously presented from the 157

isolation experiments (see Section 2). Generally much higher levels of

hyaline fungi were encountered than pigmented forms, corresponding to

the predominant fungal type of Primary Moulds at 3 months and a mixture

of Basidiomycetes and Secondary Moulds at 12 and 18 months. Low levels

of pigmented hyphae, either separately or in association with hyaline

forms, corresponded to the generally lower levels of Stainers and Soft

Rots in the untreated samples.

The situation in the treated material (Fig. 49) was not quite

so clear since at the later samplings (12 and 18 months) soft rot fungi

were dominant, almost to the exclusion of all other groups, although

from the direct observations hyaline fungi were still at relatively high

levels. It is likely that much of this corresponded to very young soft

rot fungi which lack pigment at this young age. This age structure

cannot be taken into account in the direct observations since the scoring

was a static process considering the nature of the fungus at that par-

ticular point in its life cycle only, offering none of the dynamics

available in isolation work. However, despite this disadvantage the sub-

division of colonizing hyphae yielded very useful indications, but required very careful interpretation.

From the results discussed here certain trends may be defined:-

1. Colonization and decay proceeded at a totally

comparable rate in untreated and treated birch.

CCA made no impact on inhibiting colonization

nor on inhibiting decay.

2. The fungal composition and nature of decay were=

quite different in the treated and untreated

wood. Basidiomycete attack was the most impor-

tant in the untreated material, accounting for

all but a small portion of the decay whilst soft 158

rot attack was solely responsible for the decay

of treated material.

3. The introduction of CCA made a difference at the

cellular level altering the course of fungal

succession, allowing the soft rots to flourish

whilst inhibiting the Basidiomycetes.

4. Fibres are the dominant tissue type and the

tissue most heavily colonized and attacked.

5. Vessels, together with fibres seemed to be the

main routes for colonization, rays playing a

minor role.

6. After eighteen months, fibres and rays showed

signs of fungal attack in treated and untreated

wood, whilst vessels remained unattacked in both. 159

3.4 GENERAL DISCUSSION

Two main areas of comparison are important. Firstly, intra-

treatment differences, that is, the difference between untreated pine and

untreated birch, and between treated pine and treated birch. Secondly,

the inter-treatment differences, that is, between untreated pine and

treated pine and between untreated birch and treated birch.

From the preceding discussions (Sections 3.3.1 and 3.3.2.) eight main characteristics emerge which encompass the similarities and differ-

ences between the four conditions. These characteristics can be seen

in Table 5.

The first two categories are constant and determined by the

experiment at its inception. Thus cell composition is determined by the choice of wood, tracheids being the most numerous element in softwoods whilst fibres constitute the dominant cell type in hardwoods.

The design of the experiment also fixed the levels of CCA in

the timbers. Thus, controls contained no preservative whilst the rest were treated with a 1% solution of CCA using a Bethell process which resulted in the birch giving a net dry salt retention of 7.47 ± 0.40kg m-3 and the pine 8.17 ± 0.29 kg m-3.

Since all timber blocks were randomised in an area as uniform as possible in terms of abiotic and biotic variables, and since all specimens were sampled randomly and analysed in an exactly comparable way the reasons for any similarities or differences must reside with the two fixed characteristics detailed above, namely, the nature of the substrate itself, both macroscopic and microscopic, and its response to environmental factors, and the level of preservative present.

The possible basis for any differences and their significance will be discussed in full in the next section (Section 4) and here it is proposed only to draw together the main areas highlighted by the Direct

Observational Technique. TABLE 5: SUMMARY OF COLONIZATION AND DECAY DATA

PINE BIRCH CHARACTER UNTREATED TREATED UNTREATED TREATED

1. Dominant Tissue Type Tracheids Tracheids Fibres Fibres

-3 -3 2. CCA Concentration Zero 8.17 ± 0.29 kg m Zero 7.47 ± 0.40 kg m

3. Fungal Climax Basidiomycete Soft Rot Basidiomycete Soft Rot

4. Primary Colonization Ray Parenchyma Ray Parenchyma Vessels + Fibres Vessel + Fibres Route

5. Colonization Rate Linear Same as untr. until Linear Same rate as 1 yr then decrease untreated

6. Decay Rate Slow for 1st yr No decay Linear Same rate as then rapid increase untreated

7. Basidiomycete Attack 18 months only None 12 & 18 months 3,12 & 18 months

8. Soft Rot Attack 12 & 18 months None 3,12 & 18 months 3,12 & 18 months

9. Main Decay Organisms Basidiomycetes - Basidiomycetes Soft Rots 161

Fungal composition showed a.basidiomycete climax in untreated

wood and a soft rot climax in treated wood, regardless of wood species.

This was inferred from the observations rather than proved by them as

was the case with the isolation work (Section 2.2.1.). Their presence

in the wood was not quantified, rather their effects were, that is, their

characteristic wall damage, their presence being inferred because of the

observable decay. From these qualitative observations and the quanti-

tative ones of decay it was evident that Basidiomycetes were dominant in

untreated birch and pine and were excluded from treated specimens, their

position as climax organisms being taken by soft rot fungi. This high-

lights the problems of detailing the nature of the furkal colonists from

direct observations only. The 'Hyphal Type' group could be split up

further to overcome this. For example, the 'Hyaline' category could be

further divided to include septate and aseptate hyphae. The latter

group would include some of the Primary Moulds such as the Mucorales.

The presence of clamp connections could be scored for. However, by increasing the number of options the time required for scoring becomes

prohibitively high and if it is this type of information that is of

primary importance isolation work would be more valuable and not observational work of this nature. The use of unstained, unfixed Clive')

sections and micromanipulation techniques may prove useful in this field,

but the problems are still enormous and it will be some time before individual hyphae can be removed from a scored area and given the right cultural conditions to grow and sporulate and thus be identified.

It is in detailing colonization rates and routes and the type and rate of decay that the Direct Observational Technique becomes most useful. It is comparisons of these types which have emerged from this study.

For the first year there were no significant differences in the colonization rates between any of the treatments. Thus, after one 162

year, colonization was of the same order in all four treatments. The

slight differences were probably attributable to differences in wood

moisture content. Thus, it would seem that colonization rates over the

first year are essentially the same irrespective of preservative treat-

ment (at the 1% level) and species. However, after one year significant

differences were detected.

The amount of fungus in the treated pine dropped and was

significantly lower than in any of the other treatments at eighteen

months. Levels of colonization at eighteen months in untreated birch,

treated birch and untreated pine all increased to significantly higher

values than those at one year and that of the treated pine, with no

significant difference between themselves. Thus, the presence of CCA

did not significantly affect the total levels of fungal colonists in the

birch whereas it caused a significant drop in the pine after eighteen

months, treated birch specimens acting as untreated ones as far as total

fungal colonization was concerned.

The above colonization comparisons were based on values for

all cell types, that is, total tissue colonization. However, comparisons

of the rates of colonization in specific cell types revealed that two

different pathways were involved. In the pine, both untreated and

treated, the primary route for colonization was via the ray parenchyma

and then into the axial tracheids and the rest of the cells. In the

birch the primary routes for colonization seemed to be via the vessels

and fibres and then to the other cells, untreated and treated specimens

acting in a similar manner. In order to decide between the fibres and

vessels as the primary route two sizes of section thickness would be

required (see Section 3.1.1. for full discussion) and samples at earlier

time periods would need to be scored. In order to monitor this aspect

of colonization more fully it would be advantageous to score a greater area in a totally random manner and not be constrained to the 3mm. depth 163

as was the case in the present study and also to combine some transverse,

and tangential longitudinal sections with the radial longitudinal sections

scored here.

The technique was particularly useful in monitoring decay

rates and the type of decay present. It is in this category that the

fundamental differences between the treatments were revealed. The most

notable fact was that the treated pine showed no evidence of actual decay during the first 18 months of exposure. Although fungi were present,

they were deriving no nutritional benefit from the wood itself. The

treated birch, however, showed none of this immunity from decay, it being attacked steadily from three months. At 3 months, the levels- of decay were not significantly different between the untreated and the treated birch. Decay progressed more:slowly in the untreated pine than it did in

the birch, the pine showing no decay at 3 months. At 12 months the amount of decay in the untreated pine was significantly less than in the untreated birch, there being no significant difference between the untreated and

treated birch. However, in the ensuing 6 months decay increased rapidly so that at 18 months there was no significant difference in the amount of decay in untreated birch, treated birch and untreated pine. The presence of CCA thus inhibited decay in the pine but not in the birch.

At a gross level, therefore, the presence of CCA has had no effect on the birch, in marked contrast to the pine, with neither colonization nor decay rates significantly different between treated and untreated birch. However, at a cellular level the presence of the preservative has had a significant effect on both the organisms and the nature of the observable decay. Thus, Basidiomycetes were excluded from the treated material, allowing the Soft Rots to succeed as the climax organisms. No basidiomycete attack was present in the treated birch_ whereas it was evident at both 12 and 18 months in the untreated -material. Soft rot 164

attack, however, was evident at all three sample times in both treated and untreated birch. Soft rot attack in untreated pine was restricted to 12 and 18 months and basidiomycete damage to 18 months only. Thus, although decay rates were comparable between treated birch, untreated birch and untreated pine, the nature of the decay was quite different. 165

SECTION 4 GENERAL DISCUSSION AND CONCLUSIONS 166

Two techniques have been developed to monitor the colonization and decay of wood in soil contact. The enumeration of the micro-organisms involved in the colonization/decay process was achieved firstly by their isolation onto selective, nutrient media and secondly by direct microscopic observations of the activities of the fungi at the cell wall level.

The four media used for isolations were chosen and developed to collect as much information as possible of the range of the types of colonizing fungi and their identification as well as details of the important decay fungi. This resulted in the acquisition of generalized information of colonization trends and a detailed account of the appearance and changing dominance of the decay fungi.

Progressive changes were observed in the wood with different species of micro-organisms colonizing at different times in a successional pattern.

In the untreated stakes the pine samples were consistently wetter than the birch ones sampled during the first three months of exposure.

This must have been due to differences in permeability of the two timbers since there was no significant difference between moisture content or moisture potential of the soil surrounding any of the stakes of different species at the time of sampling. This difference in wood moisture content was reflected in the number of micro-organisms isolated from the two timbers. Once introduced into the soil, the stakes took up water until an equilibrium moisture content had been reached between wood and soil.

This occurred gradually and irregularly. Over the period of the first three samples micro-organisms colonized where the moisture content was sufficiently high to allow their growth, thus exhibiting a very patchy distribution. However, bacteria were constantly isolated first and in greater numbers than fungi, showing their pioneer role in colonization, arriving with the water. Non-decay Primary Moulds and Stainers were the first fungal colonists, probably gaining access through the destruction 167

of pit membranes by the Bacteria.

Between 3 and 6 months both wood species became visibly wet at

the groundline and there was a wider distribution of micro-organisms with

the first bacteria at 40mm and the presence of the first decay fungi, the

Soft Rots. Soft Rots were isolated in low frequency after 3 months and many soft rot cavities were observed at 3mm in untreated birch, although fewer in untreated pine. Species composition was the same in both woods.

However as discussed in Section 2, the soft rot fungi were soon displaced by Basidiomycetes in both untreated timbers. The basidiomycete species composition, however, was quite different. In untreated pine Sistotrema brinkmanii comprised 93% of all the isolated Basidiomycetes. Because it showed no reaction in sawdust culture it was classified as a non-decay basidiomycete. The remaining 7% was an unknown white rot, IC/CC/BSI3, which was isolated only after 12 months. However, in the untreated birch

S. brinkmanii and a second non-decay basidiomycete, IC/CC/BS7, comprised only 12% of the isolated Basidiomycetes. The remaining 88% were all white rots. This was reflected in the Direct Observations where no basidiomycete attack was seen in untreated pine whereas 47% RPA of the birch fibres were attacked by Basidiomycetes after twelve months. The distribution of

Secondary Moulds also reflected this difference in decay with high numbers isolated from birch and very few from pine. After 18 months the non- decay Basidiomycetes had been replaced by white rots in the pine. White rots remained dominant in the birch. After 18 months, therefore, Direct

Observations revealed widespread basidiomycete attack in both pine and birch. Although soft rot fungi were present in low numbers after 12 months, being replaced by Basidiomycetes, cavities were still observed after 18 months. Whether these were relics of an earlier attack or whether active soft rot was still present could not be determined. Although no soft rot fungi were isolated after 18 months, the restricted nature of the isolations (wood veneers in damp chambers) made it impossible to say that 168

they were definitely absent. From visual observations there seemed little

direct evidence of active soft rot and it is likely that relic cavities

were being scored.

The main advantages of isolation work were that the micro-organisms

involved could be isolated and identified. These could then be tested in

culture for their decay capabilities, affect on the wood cell wall, inter-

actions between isolates and used to test the effectiveness of new pre-

servatives. All of these, particularly any screening of wood preservatives,

are much better performed using fresh isolates of organisms important in

the natural environment and not those which have been in culture for many

years. For example, on the basis of the present work any soft rot tests

would be best performed using Phialophora fastigiata and not Chaetomium globosum which historically has been the main soft rot test organism.

However, isolations have the disadvantage of being very time consuming.

Correct identification of fungi is very difficult and it cannot be assumed

that all of the fungi inhabiting the wood have been isolated. Perhaps most importantly it provides no information about the state of the organism, whether resting spore or active mycelium, or about what the organisms are doing at the cell wall level and their nutritional sources. The results of any cultural tests must be extrapolated from ideal laboratory conditions to the much more complex environment of wood in the field.

The activities of colonizing fungi at the cell wall level were detailed using a veneer-microquadrat scoring method. This Direct

Observational Technique was developed by adaptation from the analytical methods commonly employed in the ecology of higher plant communities and was a system of rapid data collection of all aspects of colonization and decay. Thus various categories of colonization and decay were scored for in microquadrats over the area of thin sections of wood. The categories used are not immutable and different ones could be substituted to suit 169

specific requirements. The data was collected as a series of coded

numbers and then fed into a computer for statistical analysis. The analysis

can be as general or specific as required by the investigator.

Direct observations overcame some of disadvantages of isolation

work in that direct evidence was provided of how the micro-organisms were

behaving in actual field conditions. When approached quantitatively rates

for colonization and decay and the type of decay could be calculated.

However, using only direct observations, the identification of the fungi

is virtually impossible, although their ecological grouping can often be

inferred from the growth and decay patterns within the wood. Methods of sampling must be carefully considered and samples should be taken objectively, from random or predetermined positions, and not subjectively from areas that appeared to lie colonized or decayed.

Direct Observations, therefore, yielded much valuable information about changes at the cell wall level. They were a numerical approach to the decay of wood and, possibly for the first time, can be analysed using rigorous statistical methods, aided by a computer, because of the large replication and the relatively straightforward way in which the data was collected. The details of the technique have been described in Section 3 and the results can be summarized here. The collected data was compiled by the computer and supplied as Relative Percentage Area of each scored character. Thus, the types of colonizing hyphae and rates of colonization were calculated together with their passage through the different tissues of the wood. All of the major types of decay were monitored from their first appearance and their relative importance in each treatment assessed.

All comparisons were tested using a two-tailed t-test producing a valid numerical expression of all observations.

Individually each technique only provided part of the information required to appreciate fully the colonization/decay process. Together the two techniques provided information as to the identity of fungi inhabiting 170

wood and their affect on the cell wall, and thus the wood as a whole.

Each technique has, in addition, substantiated, to an appreciable extent,

the findings of the other.

There were considerable similarities between the situation in the

two untreated species of wood, but great differences between colonization

and decay patterns in the treated species.

When untreated, debarked wood was introduced into the soil it be-

came wet allowing the ingress of micro-organisms from the soil population.

The wood acted as a selective screen, so that not all soil inhabitants

penetrated the wood. Those micro-organisms which could penetrate the wood

were either passive inhabitants scavenging for nutrients or true wood- destroying organisms which could Utilize the wood as a sole source of carbon and nitrogen. The colonization pattern was similar to that of trunk, branch or stump-wood in the natural environment and followed a successional pattern of:-

Bacteria

Primary Moulds

l Stainers

Soft Rots (initiation of decay)

Basidiomycetes & Secondary Moulds (bulk of the decay)

The Basidiomycetes represented the true climax organisms of the succession on untreated wood. Recent observations on the field stakes alter 31 years exposure have shown many of the untreated stakes to be in an advanced state of decay. Decay in the birch stakes was principally due to white rot and Stereum hirsutum, a white rot, was observed fruiting on the top of 171

several untreated birch stakes. However, the decay of many of the un-

treated pine stakes was due principally to brown rot. The brown rot

Poria sp. (Plate 15.1) was found fruiting on the side of an untreated

pine stake which showed signs of very advanced brown rot. However, white

rot was still present in some of the untreated pine stakes and an unknown

basidiomycete, IC/CC/BS24, was found fruiting on the front face of one

stake (Plates 15.2 & 15.3). Using Fergus's key (1960) this was identified

as Corticium sp. Plate 15.2 also shows how well the resin coating has

stood up to 31 years exposure in the soil. Thus, white rots may represent

the climax in birch and brown rots the climax in pine. White rot is

usually regarded as being more common in hardwoods than in softwoods

whereas brown rot is usually considered to be more common in softwoods

than hardwoods (Cartwright & Findlay, 1958; Rayner & Todd, 1979).

The treated pine became visibly wet at the groundline very

quickly. Associated with this was a very rapid colonization by bacteria,

showing their greatest numbers in depth after 16 days than in any other

treatment. Fungal colonists also rapidly became established, much quicker

than in the treated birch whose moisture content remained low, even after

3 months and showed little colonization.

Soft Rots were well established after 6 months and showed a

similar species composition and distribution in both treated pine and birch.

From direct observations, colonization of the treated birch gradually in-

creased to a peak after 18 months with a similar increase in the incidence

of obse.vable soft rot attack, both Type 1 and Type 2 (after Corbett, 1965).

However, in the treated pine colonization by Soft Rots increased to a

peak at 12 months and then plateaued, falling slightly by 18 months and at no time was there any evidence of soft rot attack. Thus, soft rot fungi must have derived their nutrition from sources other than the wood. Three

processes are likely to be of importance in the maintenance of the soft 172

rot fungi. Firstly, wick action causes a flow of water to pass through

the wood and this can bring with it soluble nutrients, including nitrates,

from the soil. Secondly, if the fungi maintained contact with the soil

outside the wood they could translocate soluble nutrients from the soil.

Stakes were often seen with clumps of soil adhering to the exposed face

and this may afford a stable contact with the external environment.

Thirdly, nutrients may be provided by parasitism or saprophytism of other

colonists. This was observed in untreated pine (Plates 9.1- 9.3), al-

though not in treated stakes. Carbon will probably become limiting since,

although present in excess in the wood, it is unavailable in treated pine

due to the presence of the preservative and wick action, translocation or

hyphal death will not provide a major alternative source. This lack of

available carbon may explain the decreasing levels of colonization of

treated pine after 12 months.

The wood itself, treated with CCA, must have acted as a selective

screen for colonizing fungi. High levels of copper-tolerant Soft Rots

were isolated from treated wood. These must form part of the natural soil

population since the same species were isolated from untreated wood, but

in much lower numbers. The soil forming the Lodge Site at Silwood Park

did not contain abnormally high levels of copper (see Section 1.3.3.2).

Analysis of the soil showed that it contained 16 ppm of copper and this

is well within the range of copper concentrations found in normal soils

(2 - 100 ppm) and in fact lower than the average of 20 ppm (De Haan &

Zwerman, 1978). Selection for copper-tolerance could have taken place within the soil in the vicinity of the stakes, at the wood surface, or within the wood.

Because no decay occurred in the treated pine stakes very few

Secondary Moulds were isolated. This was in contrast to the situation in treated birch where soft rot was well advanced by 12 months and there was 173

a relatively high frequency of isolation of Secondary Moulds, comprised

of only three species.

No Basidiomycetes were isolated at any time and no evidence of

basidiomycete attack was observed in any treated stakes. Thus, despite

the very low level of treatment (1% treating solution of CCA) the re-

tention of CCA was sufficiently high to prevent colonization by Basidio-

mycetes.

A recent field assessment of the stakes after 31 years in the

ground has shown no evidence of any change in this pattern. Treated pine

showed no signs of soft rot attack whereas treated birch stakes were in

the advanced stages of soft rot decay.

In CCA-treated stakes Soft Rots were the climax organisms of the

succession, becoming important only when the Basidiomycetes were excluded.

The pattern exhibited was:-

Bacteria

Primary Moulds

1 Stainers

1 Soft Rots & Secondary Moulds (initiation of decay)

It is therefore evident that separately each technique only re-

vealed part of the overall colonization/decay process whilst together one

substantiated the other and a more complete picture was obtained, not only of the progression of the organisms, but of their nature, that is,

what they were doing to the wood and to each other.

A number of important results can be obtained when both Isolations and Direct Observations are employed. This is because the experiments can 174

be set-up on a sound statistical basis which makes it possible to analyse

the results and information collected quantitatively to give reliable data. This yields a number of important findings:-

a) the organisms present,

b) their relative importance,

c) their progression and/or succession,

d) what the organisms are doing to the wood,

e) what the organisms are doing to each other,

f) what state the organisms are present in the wood.

From these the major conclusions are:-

a) that the two techniques together are better than

either one separately,

b) the importance of identifying ecological groups

rather than pure taxonomy,

c) that both Isolation and Direct Observation can be

meaningfully enumerated to give a numerical ex-

pression of the results of each technique.

Many attempts have been made and will be made to enumerate the colonization/decay process in wood but what cannot be resolved is the main problem that it is impossible to see what is happening at the time at which it occurs. By employing the two techniques described here a major step-forward has been achieved. Much has still to be discovered but the philosophy outlined here will help to answer a number of questions and build-up a more accurate picture of the sequence of events as they occur. 175

SECTION 5: IDENTIFICATION OF FUNGI FROM FIELD STAKES 176

5,1 INTRODUCTION

The isolation and identification of fungi from wood is a subject

that should not be undertaken lightly. Any project involving this aspect

of biodeterioration must be very carefully planned since, during a series

of samplings, the number of isolates which must be *processed increases

almost exponentially with a similar increase in the time commitment.

In systematic mycology there is a vast literature which needs to

be considered. For accurate identification microscopic characters have

to be considered for all species. A truly systematic approach to wood

mycology thus requires enormous resources in terms of man—hours. Thus,

during a project, part of which involves the isolation and identification

of micro—organisms, only a relatively short period of time is available

and therefore compromises must be made in the procedures to be undertaken.

During the present project samples were taken at 2, 4, 8, 16,

32, 96, I84, 265 and 363 days. At each time, with the exception of the

first three where treated stakes were not supplied, isolations were made

from 3 stakes, from each of 4 treatments. From each of these 12 stakes

isolations were taken at 3 depths. At each depth 32 inoculum splints were divided between 4 different media (4 splints per plate). Thus each sample produced 288 agar plates containing a total of 1152 inoculum splints. Each inoculum could produce 4 fungi, though it was rarely more than 2. Thus, on an average of 2 fungi per inoculum, each sample could yield over 2000 fungal isolates. In practice the number was considerably smaller than this, especially in the early stages, since many inocula were sterile and many produced only one fungus. Despite this approximately

1000 isolates per sample was not uncommon in the later stages--(1007 fungi were isolated at 363 days). Purification of the isolates can be a lengthy process involving several cultural transfers leaving less time for identification. In order to rationalise the number of cultures subjective decisions have to be made about which isolates are the same. This often 177

has to be made at a gross level by looking at the plate culture, its

form, colour and habit, since time does not allow each isolate to be

examined microscopically or after slide cultures had been prepared.

These decisions became easier with experience and familiarity with the organisms. Thus a small number of type variants were retained for more detailed, microscopic observations and specific identification. Much of

the detail of fungal distribution was, therefore, based on these visual, cultural characteristics. The detailed identification based on type species was then extended to the type variants previously recorded as a code name. For example, although Phialophora fastigiata occurred a total of 469 times during the first year's sampling its identity was confirmed microscopically less than 100 times, that is 1 in 4-5 times. For some

'less important' species the ratio may have been greater. 178

5.2 MATERIALS AND METHODS

Once the isolates had been purified (Section 2) attempts were

made to identify them. Based purely on the visual characters of pigment-

ation, growth rate, form and texture all isolates were assigned to one

of four groups, for convenience of handling:

Group 1 - Bacteria and Actinomycete - like organisms.

Group 2 - Fast growing, non-pigmented or exotically

coloured, 'mould-type' fungi.

Group 3 - Stainer-type fungi with pigmented hyphae

ranging from dark blue to black.

Group 4 - Basidiomycete fungi.

Bacterial cultures were initially separated on the basis of

their Gram stain reaction. Although no real attempt was made to classify

the bacteria any further, some tests were performed as suggested by

DeGroot and Johnson (1976) for the separation of species of Bacillus. As

described earlier most of the bacterial isolates were Gram-positive rods

of the Bacillus-type as illustrated in Plate 4.1.

It occasAonally proved difficult to separate the fungal com-

ponent of a mixed culture of fungus and bacteria, or yeast. To free the

fungus from the bacteria the Van Tiegham Ring Method was used (Raper, 1937).

This consisted of a small glass ring, fifteen millimeters diameter, with

three small glass beads fused to its lower surface. Agar was poured into

a petri dish containing one or more sterilized rings. A portion of the

mixed culture was transferred to the agar in the centre of the ring where

the fungus was able to grow through the agar, below the ring, to the

surrounding medium, emerging free from bacteria, or yeast, which were unable to grow through the agar. This method proved to be particularly useful when the fungal component produced very little aerial mycelium.

For all fungi assigned to groups 1 and 2 identification was based on the nature of the sporing apparatus. With the exception of three

179

Ascomycetes which showed ascocarp development in culture, this repre-

sented the development of asexual spores, either sporangiospores or conidia. In some cases it was possible to mount sporing structures directly from the culture, for observation under the light microscope.

However, spores are designed for dispersal, consequently disturbance caused by mounting often obscured the more subtle structures on which reliable identification was based. It was, therefore, necessary to set—up slide cultures in order to observe the asexual fructifications

(Figure 51) .

FIGURE 51: Damp Chamber for Slide Cultures

Sterile glass slide

No. 0 cover glass

Inoculum plug PLAN

SECTION Petri dish

Glass supporting Several layers of damp rod filter paper (to maintain humidity) 180

For each unknown fungus an agar plug (5mm. diameter) was removed

from the actively growing hyphal front and placed onto a sterile glass

slide in a sterile damp chamber (Figure 51). A sterile, circular, number

nought, cover glass was placed on each agar plug. The whole chamber was

then incubated in the dark at 22°C. During the incubation mycelium grew

from the agar plug across the cover glass where it was induced to spore

due to the low nutritional status. This was observed directly under a

stereo microscope and at the appropriate stage the cover glass was re-

moved from the chamber, free of its agar plug. The mycelium and any

fructifications were fixed to the cover glass by immersion in absolute

alcohol and then stained in either aniline blue in lactic acid or lacto-

fuschin. The stains were made up to the following concentrations (w/v):

1. Aniline Blue : 0.1 g. Lactic Acid 100 ml.

2. Acid Fuschin . 0.1 g. Lactic Acid 100 ml.

In cases where cell wall detail alone was required the fungi

were stained in Ammoniacal Congo Red of the following composition (w/v):

Congo Red 10 g. Ammonium Hydroxide (specific gravity 0.88) : 5 drops Distilled Water : 100 ml.

For many of the Dematiaceous Hyphomycetes staining was superfluous since

adequate contrast was achieved by the natural wall pigments. In these

cases fungi were mounted in lactic acid. These temporary mounts were

made semi—permanent by sealing the edges with Gurr's Glyceel. These

preparations have been found to last well, retaining detail for two years

or more if stored flat. Ordinary (colourless) finger nail varnish is

also suitable.

The use of lactic acid was favoured over that of lactophenol, often quoted as the standard mountant. The main reason for this is that

the presence of phenol can cause some shrinkage of the structures and 181

also act as a clearing agent, thus making penetration of the stain more

difficult.

Due to the number of isolates involved, which ran into thousands,

slide cultures were not made of all isolates. This would be the ideal

situation because of the visual similarities of many closely related, but

distinctly separate species, especially amongst the stainers, and for

confident identification to the species—level. In this situation exper-

ience and personal judgement have to be used to group similar isolates

together. Representative slide cultures can then be made to confirm

identification.

Carey (1980) has suggested the use of three comparative media

to separate different types of stainers isolated from window joinery.

The same behaviour on each medium, Malt, Czapek—Dox and Cornmeal Agar,

indicated an identical fungus. This was particularly useful in grouping

Aureobasidium pullulans isolated from window joints into different strains.

This technique has proved valuable in the present study, but due to the

relative paucity of stainers, particularly A. pullulans, in ground contact

material when compared with wood out of ground contact, it was not used

extensively.

The more common species and those with particularly diagnostic

cultural characteristics were identified with a high degree of certainty

without making slide cultures.

Initial identifications were made using the numerous keys avail-

able. For the Dematiaceous Hyphomycetes, of which many were isolated

from wood, the keys produced by Ellis (1971, 1976) are authoritative. For

general microfungi, Barnett and Hunter (1972), Von Arx (1974),=Barron

(1972) and Domsch and Gams (1972) were found to be particularly useful.

The taxonomic keys and cultural information contained within The Fungi:

An Advanced Treatise, edited by Ainsworth, Sparrow and Sussman (1973) were

also invaluable. 182

Representatives of all isolates of the 'mould' (Group 1) and

'stainer' (Group 2) types were taken to the Commonwealth Mycological

Institute for authorisation of all identifications. Time was spent at

the Institute in order to identify all isolates as far as possible, with

help from the specialist mycologists working there.

Several isolates, particularly some of the Dematiaceous Hypho-

mycetes, had remained sterile in culture thus preventing identification.

Some of these were induced to spore after a 'Black-Light' treatment.

The importance of the light-factor in inducing sporulation in fungal

cultures has been demonstrated by several workers, particularly Leach

(1961, 1962a, 1962b, 1963). The wavelengths found most effective in in-

ducing sporulation are in the near ultra-violet region of the spectrum

(known as 'Black-Light'). Leach recommended an alternating cycle of

12 hours UV and 12 hours darkness in order to accommodate any fungi

requiring a dark period. Non-sporing isolates were streaked onto a suitable solid medium in a plastic (polystyrene) petri dish and incubated. in

the dark at 24°C for 2-4 days. Glass petri dishes are unsuitable because

the transmission of near ultraviolet by glass is very poor. After this

period of growth the isolates were placed under fluorescent lamps. The

light bench consisted of three fluorescent tubes 6" apart. The centre

light was a Phillips near ultraviolet 'Black Light' tube TL 40w/08 and

either side of this was a daylight tube, Phillips cool white MCFE 40w/33.

All three were controlled by one time switch set on a 12 hour on/off cycle. The isolates were placed 14" below the fluorescent tubes and the dishes were sealed with Scotch sealing tape to prevent the culture medium drying-out. Some cultures required six weeks under this regime for adequate sporulation, four weeks being the standard induction time.

Dematiaceous Hyphomycetes and Coelomycetes responded particularly well to black light treatment. Fresh isolates also responded much better than isolates that had been in culture for a length of time and undergone several transfers. 183

Basidiomycetes were much more difficult to identify. Reliable

identification should be based on the structure of the basidiocarp which

was difficult to induce in culture.

The presence of clamp-connections was indicative of a Basidio-

mycete but since clamps are produced only by the dikaryon and not during

the monokaryotic phase of the life cycle, absence of clamps in an isolate

does not preclude it being a Basidiomycete. All cultures possessing

clamps or looking culturally like a Basidiomycete (white, slow growing,

slightly fluffy culture growing on benomyl agar and possibly bleaching

agar) were inoculated onto a supplemented sawdust medium in order to

determine their wood decay characteristics. The medium was based on that

used at the Princes Risborough Laboratory (Carey, 1975), modified after

Badcock (1941).

Scots pine sawdust was sieved through a 2mm. mesh and supple-

mented with 3% maize meal and 2% bone meal. Distilled water was added

until the sawdust particles readily cohered, forming large clumps, when

squeezed together. At this point the moisture content should be between

200 - 300% (Carey, 1975). The sawdust medium was packed into large

boiling tubes, 33mm. internal diameter. Each tube was fitted with a

cotton wool bung andautoclave sterilized for twenty minutes at 20 psi.

Individual sawdust tubes were inoculated with the test fungus and incuba-

tedinthē dark at 22°C for two weeks, observations being regularly made

for any colour changes. Brown rot fungi gradually darkened the sawdust,

due to the selective removal of cellulose, leaving the lignin as a

brown, friable residue. White rots initially formed a rich brown coloured

zone which moved away from the point of inoculation with growth of the

fungus and was followed by bleaching of the culture medium as both lignin

and cellulose were removed. Non-decay Basidiomycetes left the sawdust unaltered. In this way the test fungus was shown to be a white rot, brown rot or non-decay Basidiomycete. 184

Once their decay characteristics had been established attempts were made to induce fructification to allow identification. After the sawdust had been completely colonized the cultures were brought into a diffuse, natural light, direct sunlight and strong artificial light being avoided. The sterile cotton wool plug was replaced by a moist square of cotton wool placed along the inner face of the tube in contact with the edge of the sawdust to encourage colonization of the cotton wool, which was kept moist by periodic watering. The tubes were lain almost flat and any changes in orientation avoided (Plate 14.1). Badcock (1943) noted fruiting time to be anything up to 31 weeks after inoculation. 185

5.3 RESULTS

Wherever possible, identification was based on the nature of

the sporing apparatus. Section 2.2.2. described the grouping of isolates into Ecological Groups and although similar methods were employed for

the identification of all isolates for convenience they will be considered here within those Ecological Groups.

5.3.1. PRIMARY MOULDS

With the exception of three species, the Primary Moulds were all

Hyphomycetes (s.d. Deuteromycotina). The three exceptions were all

Zygomycetes (s.d. Zygomycotina), members- of the order Mucorales. These fungi readily sporulated in culture producing asexual sporangia which were identified using the key produced by Zycha et al. (1969). In this way Mucor hiemalis Wehmer, Mucor plumbeus Bon. and Zygorhynchus heterogamus Vuill. were identified as Primary Moulds.

The Hyphomycetes were more difficult to identify since sporulation in culture often required very specific conditions (e.g. black light, cold period, diffuse daylight). Thus, several species which were isolated remained sterile and therefore unidentified as Mycelia Sterilia

(Hyaline). Several of those which were identified were isolated only infrequently and irregularly distributed. However, three genera were regular members of the flora; Verticillium, Penicillium and Fusarium.

Verticillium and allied genera (Cephalosporium, Acremonium and related genera producing hyaline phialospores) have been the subject of a major taxonomic revision by Gams (1971) and reference should be made to his book for complete details. Verticillium is characterized by the production of whorls of phialides (verticils) of equal size inserted at the same level below the septum (Plate 10.1). However, conidiophore development is not always typical. In many cases phialides arise singly or in pairs (Plate 10.2) and isolates may be mistaken for Acremonium. 186

PLATE 10

Primary and Secondary Moulds

All specimens are from slide cultures and stained with cotton blue in lactic acid.

10.1 & 10.2 Verticillium lecanii (culture no. IC/CC/M9). Primary Mould.

10.1 Typical sporulation with phialides borne in verticils.

10.2 Acremonium—stage with phialides borne singly or in pairs, arising

laterally.

10.3 & 10.4 Penicillium cyclopium (culture no. IC/CC/M6). Primary Mould.

Range of conidiophore morphology.

10.3 Single branched conidiophore.

10.4 Simple monoverticillate penicillus.

10.5 & 10.6 Glioeladiun roseum (culture no. IC/CC/M54). Secondary Mould.

10.5 Verticillium state with phialides borne in whorls, an early stage

in conidiogenesis.

10.6 Characteristic penicillate habit of mature conidiophores with

appressed phialides. At maturity spores collect as a wet spore

cluster above phialides. .4

10.3 17 PM 10.4 13 pm 188

Penicillium species, regular members, particularly of the treated

wood flora, were identified on the basis of the classification by Raper

and Thom (1949). They divided Penicillium into four major sections:

Monoverticillata, Asymmetrica, Biverticillata-Symmetrica, and Polyverti-

cillata. This division was based on the pattern and complexity of the

conidial structure or penicillus. The larger portion of the genus is

contained within the Asymmetrica although intergradation occurs between

the major sections as shown in Plates 10.3 & 10.4. Plate 10.3 shows the

more typical single branched conidiophore of P. cycZopium Westling

(Asymmetrica). Plate 10.4 shows a simple monoverticillate penicillus

from the same species.

Identification of the Fusarium species was based on single spore

isolates as described by Booth (1971b, 1977).

5.3.2 STAINERS

Taxonomically the Stainers were members of three classes: the

Pyrenomycetes (sod. Ascomycotina), the Coelomycetes and Hyphomycetes

(both s.d. Deuteromycotina). Three ascomycete species were isolated, two

of which produced fertile perithecia in culture and were identified using

Dennis's Key (1978) as Ceratocystis sp. and Coniochaeta subcorticalis Link.

C. subcorticalis (Plates 11.3 & 11.4) was of particular interest since it

is a perfect stage of the Phialophoras. Distinction between species of

Coniochaeta is based on the size and shape of the ascospores which can

be unreliable in perithecia produced in culture (Sivanesan, Pers. Comm.).

The third ascomycete, Xylaria sp., produced only abortive, horn-shaped, ascocarp primordia and was identified from its cultural characteristics and effect on sawdust. In culture it produced only appressed aerial hyphae which became encrusted, forming a tough mat over the surface of the agar. It also produced deep, black zone lines in the agar. On sawdust it produced a slow, gradual white rot reaction. 189

PLATE 11

Stainers and Soft Rots

11.1 Phoma funeti (culture no. IC/CC/S69). Stainer.

Squash preparation stained in lactofuschin. The mature pycnidium

has been split causing release of the spores (s). A young pycnidial

primordium is also evident (p).

11.2 Epicoccum purpurascens (culture no. IC/CC/S45). Cellulolytic Stainer.

Direct mount stained with cotton blue in lactic acid. Developing

and mature, muriform spores borne singly on a short conidiophore.

11.3 & 11.4 Coniochaeta subcorticalis (culture no. IC/CC/S55). Cellulo—

lytic Stainer.

11.3 Unstained section of a perithecium containing two mature asci. Dark

setae (s) which surround the ostiole are also evident.

11.4 Perithecial squash showing protective setae and several mature asci.

Unstained.

11.5 & 11.6 Phialophore fastigiata (culture no. IC/CC/S2). Soft Rot.

Slide cultures stained with cotton blue in lactic acid. Variation

of phialide morphology within species group.

11.5 Single and branched phialides, with prominent collarettes borne

terminally on the hypha.

11.6 Branched phialide borne laterally on the hypha. 11.3

•

q•

6 prn 11.6 6PM 191

Three species of Coelomycetes produced fertile pycnidia in

culture allowing identification (Grove, 1935, 1937). These were Phoma

fimeti P. Brunard (Plate 11.1), Coniothyrium fuckelii Sacc. and

Botryodiplodia theobramae Pat.

Identification of all Dematiaceous Hyphomycetes was based on

Ellis's Keys (Ellis, 1971, 1976). Most of these were isolated irregularly

and in low numbers with the exception of Epicoccum purpurascens Ehrenb ex.

Schlecht. This was very widespread and characterized by the production

of black sporodochia in culture. These consisted of dense clusters of

short conidiophores bearing muriform conidia with irregularly roughened

walls (Plate 11.2).

These fungi were classified as Stainers due to the pigmentation

of their hyphae and lack of any visual signs of soft rot activity in

inoculum splints. However, Epicoccum and Phoma have been reported as

causing soft rot under certain circumstances (Carey, 1980; Henningsson &

Nilsson, 1976a; Seehan et al., 1975).

5.3.3 SOFT ROTS

All the Soft Rots isolated were Dematiaceous Hyphomycetes, al-

though most are the anamorph (imperfect stage) of an ascomycete telemorph

(perfect stage) (Luttrell., 1979).

Ellis's Keys were again used, except for the genus Phialophora.

There was a wide variation in phialide structure making speciation diffi-

cult. The genus was revised by Schol-Schwarz in 1970 and six wood-

inhabiting species dealt with, taxonomically, by Cole and Kendrick (1973).

Schol-Schwarz grouped similar species with closely related perfect stages

into species groups. Using her classification the most numerous isolates

were from the Phialophora fastigiata group which included Ph. fastigiata (Lagerb.& Melin) Conant and Ph. melinii (Nannf.) Conant. The group is

characterized by having greyish, brown mycelium, a cottony or velvety

appearance and possessing hyphal strands. The structure of the phialide 192

was variable in Ph. fastigiata occurring laterally or terminally on the

hypha and arising singly (Plate 11.5) or branched (Plate 11,6), The main

difference between the two species was that Ph. melinii tended to produce

more aerial mycelium and predominantly single phialides often from the

peripheral hyphae of hyphal strands. Ph. sp. 3 (Plate 12.1) and Ph. sp. 4

(Plate 12.2) are best placed in the Ph. hoffmanii group. Neither could

be speciated with certainty but both were pale, cream-coloured cultures

with short, stout, swollen phialides which are characteristic of the A.

hoffmanii group. Ph. sp. 4 consistently produced asymmetrical flask-

shaped phialides (Plate 12.2). A fifth species, Ph. sp. 2, isolated only

from treated wood, possessed large, irregular clusters of short, stout

phialides (Plate 12.3).

Four other soft rot species were isolated. TrichocZadium opacum ~ (Corda) Hues was characterized by the solitary cordia which were 3 - 4

septate, smooth-walled becoming dark brown with age (Plate 12.4). As well

as being consistently isolated from treated wood it has been observed in a

sporing condition in soft rotted, treated birch fibres (Plates 12.5 &

12.6). A similar situation occurred with Humicola fuscoatra Traaen

(Plate 13.1) which was commonly isolated from treated wood and which has been observed in the wood itself, although no evidence of soft rot was

seen (Plate 13.2). H. fuscoatra was characterized by the dark, spherical

to ovoid, conidia borne singly an short conidiophores. It was distinguished by the smaller conidia (6-9 x 12pm) than those of its close relative

H. grisea (12pm diameter), a commonly reported soft rot of treated wood.

Drechslera dematioidea (Bubak & Wroblewski) Subram, was the only soft rot which was benomyl tolerant, showing strong growth on:the benomyl/ streptomycin medium. The colonies were brown or blackish brown, often velvety with irregular sclerotia forming in culture. The conidiophores were long and straight (usually 60 - 150pm) bearing clusters of clavate conidia which developed sympodially, leaving a prominent scar (hilum) when 193

PLATE 12

Soft Rots

12.1 Phialophora sp. 3 (culture no. IC/CC/S34).

Slide culture stained with cotton blue in lactic acid. The phialides

are short, stout with a swollen base and no true collarette.

12.2 Phialophora sp. 4 (culture no. IC/CC/S9).

Slide culture stained with cotton blue in lactic acid. The phialides

are flask-shaped (urniform) with a' long neck and insigificant collar-

ette.

12.3 Phialophora sp. 2 (culture no. IC/CC/S68).

Direct mount stained with cotton blue in lactic acid. The phialides

are produced in irregular clusters.

12.4 Trichocladiwn opacwn (culture no. IC/CC/S44).

Direct mount, unstained. Conidia arise singly and are heavily pig-

mented often obscuring the 3-4 septa.

12.5 & 12.6 Radial longitudinal section from treated birch stake sampled

after 1 year. Stained with safranin and picro-aniline blue.

12.5 T. opacum sporing in its soft rotted fibre.

12.6 Heavily soft rotted fibres showing evidence of the most likely

causal organism, T. opacwn. 4 • • 4040054.---4 410rim_04 -

rt 00 01 • it '41,1.0) • VW' 4111"11.

12.1 4 pal 195

PLATE 13

Soft Rots

13.1 Humicola fuscoatra (culture no. IC/CC/S8).

Unstained, direct mount showing the characteristic conidia borne

singly on short, lateral conidiophores.

13.2 Humicola fuscoatra sporing in a fibre of untreated birch. The

section (RLS) shows no sign of decay.

13.3 & 13.4 Drechslera dematioidea (culture no. IC/CC/S4),

13.3 Direct mount stained with cotton blue in lactic acid showing the

long conidiophores bearing clusters of spores.

13.4 Direct mount stained in trypan blue to show up the presence of

pseudosepta in the spores.

I3.5 & 13.6 Unknown Soft Rot, culture no. IC/CC/MSD7.

13.5 Unstained, direct mount of submerged hyphae showing microsclerotia

(s).

13.6 Direct mount of aerial mycelium stained with cotton blue in lactic

acid showing arthrosporic state. •

13.1 14 pm. 13.2 iii 14 pm.

13.6 4 197

discharged. The conidia had 2 - 7 (usually 3 - 4) pseudosepta (Plates

13.3 & 13.4).

The final soft rot, IC/CC/MSD5, remained unidentified and was

most unusual. Its initial growth in culture was a greeny-brown, cottony

to velvety mat producing numerous microsclerotia (Plate 13.5). The

centre then developed a white cottony growth which produced hyaline

arthrospores (Plate 13.6). It was initially thought to be a mixed cul-

ture, but all attempts to separate it, both at I.C. and C.M.I., failed

to do so, leaving one to suspect a pure, if somewhat unusual culture.

5.3.4 BASIDIOMYCETES

As described in Section 5.1 all basidiomycete-like cultures were inoculated onto a supplemented sawdust medium to try and induce fruit- ! ing (Plate 14.1). Two cultures responded to some extent to this treatment.

Culture No. IC/CC/BS8 spored profusely with the production of a very loose, granular fruitbody. This was identified as Sisto trema brinkmanii

(Bres.) J. Erikss. using Christiansen's Key (Christiansen, 1960). This was confirmed by Carey (Pers. Comm.) and matched with isolates from window joinery (Carey, 1980). S. brinlonanii exhibited a wide variation in culture. Some isolates produced appressed aerial mycelium with large aggregates of moniliod hyphae: hyphae with swellings (or bulbils) in chains and usually simple clamps between the bulbils (Plate 14.2). Other isolates had a relatively fast growth rate and produced white aerial mycelium which became granular in patches due to the production of a very loose fruitbody of basidia with eight basidiospores (Plate 14,3).

Culture No. IC/CC/BS16 produced a small, pale brown resupinate fruitbody, 2cm, wide. This showed some zonation, with a white margin to the fruitbody with minute pores on the undersurface. Although no spores were evident, preventing definitive identification, the fruitbody resembled that of Bjerkandera adusta (Willd. ex Fr.) Karst. This was confirmed from cultural characteristics by Rayner (Pers. Comm.). 198

PLATE 14

Basidiomycetes

14.1 Various Basidiomycetes growing on supplemented sawdust medium.

Attempt to induce fruiting on moistened cotton wool pad placed in

contact with the fungus.

14.2 & 14.3 Sistotrema brinkmanii (culture no. IC/CC/BS8).

14.2 Direct mount stained with lactofusch.in showing the moniloid hyphae

with clamp connections (c) between etch bulbilous cell (b).

14.3 Direct mount stained with ammoniacal congo red showing the loose

arrangement of the fruitbody (f) and a single basidium (b) bearing

8 basidiospores (1 out of focus and 1 dislodged). t 200

No other isolates fruited in culture. However, two others were

identified on the basis of cultural characteristics (Rayner, 1978, Pers.

Comm.). Culture No. IC/CC/BS10 with its whorled clamps on the wider

hyphae and yellow-orange pigmentation was identified as Stereum. hirsutum

(Willd. ex Fr.) Fr. Culture No. IC/CC/BS6 grew very fast, initially mostly as submerged mycelium, aerial hyphae developing later. The submerged hyphae at the edge of the colony did not bear clamps, but the aerial hyphae did (notatocoenocytic). With age the cultures acquired a salmon-pink to orange coloration and the aerial hyphae produced club- shaped cystidioles which became covered in liquid globules. From this it was identified as Phiebia merismoides Fr.

Attempts were continued to identify the unknown cultures by using some of the qultural tests suggested by Stalpers (1978).

5.3.5 SECONDARY MOULDS

Three fungi were classed as Secondary Moulds, all being Hypho- mycetes. These were Trichoderma viride sensu Bisby, Acremonium strictuur

W. Gams spec. nov. and Gliocladium roseum Bain. All three species were cellulolytic and in culture cleared cellulose medium very quickly. They were all closely related to each other and showed a teleomorph relationship with the family Hypocreaceae (class Pyrenomycetes, s.d. Ascomycotina).

G. roseum has been connected to its perfect state, Nectria g iociadioides by Smalley & Hansen (1957). Acremonium also has a Nectria teleomorph

(Luttrell, 1979) and Trichoderma has an Hypocrea teleomorph (Rifai, 1969).

T. viride was identified on the basis of Bisby's concept of a single species (Bisby, 1939) rather than using Rifai's revision into nine species

(Rifai, 1969). A. strictum and G. roseum were both identified using

Gams's Key (Gams, 1971). During conidiogenesis Gliocladium showed an acremonium state (phialides mostly borne singly) and also a verticillium state (Plate 10.5) before showing the characteristic penicillate habit of conidiophores with appressed phialides (Plate 10.6). 201

PLATE 15

15.1 Poria sp. fruitbody on the side of an untreated Scots pine stake.

The stake, which had been in the ground for 32 years, showed

signs of very advanced brown rot. The fruitbody has large, ir-

regular pores.

(Twice actual size).

15..2 An untreated Scots pine stake sampled after 31 years. The stake

showed advanced decay by white rot. On the exposed face above

the groundline (g) is the basidiocarp of an unknown basidiomycete,

IC/CC/BS24, (b).

The resin coating, even after 31 years, is still intact apart

from some weathering on the above ground portion. The resin above

ground has also darkened: (possibly the result of exposure to

ultra-violet light).

(Scale: each small division equals 1mm.).

15.3 As 15.2, but close-up of the fruitbody of basidiomycete IC/CC/BS24.

The basidiocarp is entirely resupinate and shows the character-

istics of Corticium (Persoon) Fries. The species was not deter-

mined. The loosening of the basidiocarp and slight blistering.in

the centre is due to drying.

(Scale: Imm divisions).

203

5.4 DISCUSSION

From this brief rgsumg of methodology it can be seen that the

identification of fungi is based on very exacting criteria. Correctly

applied, it becomes a very painstaking and time consuming operation for

the accurate identification of an unknown isolate and when many different

isolates are involved (up to 1000, see Section 5.1), over a large number

of samples, as is the case in any decomposition studies, time becomes very limiting. However, with careful planning and fine judgement it has

been possible to characterize many individuals and although some mistakes may have been made the main organisms involved in the colonization/decay

process have been identified with a great deal of confidence, providing much valuable information. Therefore, although many subjective decisions have had to be made, being informed decisions,a high degree of accuracy can be expected. So long as the study is approached systematically, familiarity with the organisms is soon achieved resulting in the correct identification. 204

APPENDIX 1

Layout of the Field Site

The field site consisted of 25 rows containing 20 stakes per row.

Each stake was given a treatment code and number which was marked on the side of the stake.

BrU — Untreated Birch Br — Treated Birch SpU — Untreated Pine Sp — Treated Pine

Each stake was assigned a position in the site with the use of random number tables. 1 2 3 4 5 6 7. 8 9 10 11 12 13 14 15 1617 18 1920 1 Sp SpU SpU Sp Br SpU Sp SpU SpU Br Br Br SpU SpU SpU Sp 30 99 80 108 93 101 88 70 96 31 62 116 23 131 20 75 2 SpU Sp SpU Br Sp Sp Sp SPU Sp Br 125 37 58 128 52 41. 101 85 78 88 Sp SpU Sp Br Sp SpU Br Br Sp SpU SpU Sp Sp SpU Br Sp Br 3 12 28 99 52 82 63 83 12. 51 109 74 80 19 30 8 125 58 Br Br SpU SpU - SpU SpU SpU Br Br Sp SpU Sp Br SpU / 87 70 29 66 77 107 111 14 17 117 114 33 28 21

Sp Br Sp Br SpU SpU SpU Sp SP Br SP SP Br 5 48 40 94 71 45 46 27 93 68 73 1 26 119

Br SP SP Br SP Br SpU Br SpU Br SPU Br Br SpU Br Br 6 85 85 63 25 3 63 86 9 31 89 34 61 67 10 59 20 R Br SpU Br Br SpU : SpU Sp Br SpU Br Sp Br SpU SP SpU 7 ^8 112 122 5 67 57 123 84 40 15 66 64 6 21 104 0 Br SpU SpU Br Sp Br Br Br SpU SpU Br Sp Sp SpU Sp Br SpU '• SpU SpU 8 55 81 51 125 79 32 38 66 64 38 101 131 54 7 70 95 116 83 22 W Br Sp Sp SpU Sp SpU Sp Sp Br SpU SpU Sp 9 11134 115 15 31 47 39 113 104 56 39 98 90 0 S Sp SpU SpU Sp Sp Sp Sp Sp Sp Br SpU SpU Br SpU Sp Br Sp 10 7 54 100 72 112 92 87 28 74 23 18 44 30 32 96 54 11 Ar Sp Ar Br B r Br Sp SpU SpU B SpU Sp SpU SpU Sp Sp Br 11 75 35 108 123 4 2 112 4D 79 13 10r 102 116 62 72 124 4 110 Sp Sp SpU Br Sp Br SpU SpU SpU Br . Br SpU SpU Sp Br Sp 1 2 77 43 124 86 36 19 71 5 88 90 114 76 93 57 27 59 Sp SpU Sp Br Sp Br SpU SpU Sp SpU SpU SpU SpU Br Br 13 5 82 23 65 102 18 117 56 10 53 105 91 126 98 125 Br SP Br Br Br SpU Sp Br SpU SpU Br Ar Sp SpU Br Sp 14 72 128 117 74 12 0 95 22 75 36 60 81 55 42 7 81 1 Br Br Pr Br Sp Sp Br Sp Br Br Sp Br SpU Sp 5 56 50 1J5 o 100 98 96 6 47 21 64 53 17 107 Sp Br SpU Sp SpU Sp Sp SpU SpU Sp Sp Br Br Br 16 118 32 29 52 118 89 60 67 103 130 103 113 97 11 34 Br Sp SpU Sp SpU SpU SpU Sp SpU Br Sp SpU Sp Sp 1 7 69 47 87 129 55 43 15 34 35 44 133 2 17 18 SpU Br Sp SpU SpU Sp Sp Sp SpU Sp SpU Br Sp Br Br 18 108 15 25 92 39 41 132 89 78 127 41 13 13 24 124 Sp Sp Br Br . Sp Sp Br Sp SpU Sp Sp SpU SpU SpU Sp Br Sp 19J 97 130 62 68 8 53 .6 71 84 62 90 19 94 37 126 25 42 ; Br SpU Sp Sp Sp Sp SpU SpU SpU Sp Sp Sp Sp Br Sp Sp 2 O 110 45 49 97 76 109 106 75 48 8 110 46 9 16 49 35 Br SpU Br SpU Sp SpU SpU Br Sp SpU Br SpU Sp Br Sp 21 20 106 106 77 3 2 4 9 113 11 99 49 58 79 29 2 Br Ar Sp Br SpU SpU Sp Br SpU Sp Br Sp Br 2 103 127 83 123 118 14 50 91 50 105 57 84 92 Br SpU Sp Br SpU Br Sp Br Sp Br Sp SpU Br SpU Br r , 23 Br 94 51 68 86 48 95 100 27 46 38 111 91 73 45 123 43 9 e 2, Br SpU Br SpU SpU SpU Br Br SpU Br SpU Br SpU Hr, SpU Li 33 128 109 24 60 133 102 37 129 107 2 5 104 59 1:X 127 SpU Rr SpU --'r Sp SpU Br SpU SpU Sp SpU Sp 25 26 3b 132 14 12 80 33 1 22 16 24 206

APPENDIX 2

Figures for the Percentage Frequency of Isolation of all the Ecological

Groups during the first year of colonization. The figures are rounded

to the nearest whole number and calculated on the basis outlined in

Section 2.2.3 (Page 80).

Percentage Frequency of Isolation - Positive Isolations x 100 Total Possible

The total possible isolations were:

16 for Bacteria (B) 16 for Primary Moulds (PM) 16 for Stainers (S) 16 for Secondary Moulds (SM) 24 for Soft Rots (SR) 8 for Basidiomycetes (BS)

These figures for the Percentage Frequency of Isolation were then used to derive Figures 5-20 (pages 83-100). 207

PERCENTAGE FREQUENCY OF ISOLATION

3mm Depth 10mm Depth

ECOLOGICAL GROUP B PM S SR BS SM B PM S SR BS SM

BRU64 0 0 0 0 0 0 0 0 0 0 0 0 0 2 Untreated BRU112 19 0 0 0 0 0 0 0 0 0 0 Birch D BRU40 38 6 0 0 0 0 0 0 0 0 0 0 A Y MEAN 19 2 0 0 0 0 0 0 0 0 0 0

S SPU6 880 0, 0 0 0 6 0 0 0 0 0 A M Untreated SPU22 63 0 0 0 0 0 0 0 0 0 0 0 P Pine L SPU28 13 31 25 0 0 0 0 0 0 0 0 0 E S MEAN 54 10 8 0 0 0 2 0 0 0 0 0

BRU23 6 13 0 0 0 0 6 0 0 0 0 0 4 BRU80 Untreated 63 19 .0 0 0 0 38 0 0 0 0 0 D Birch BRU11 88 A 6 13 0 0 0 0 0 0 0 0 0 Y MEAN 52 13 4 0 0 0 15 0 0 0 0 0 S A SPU104 69 25 19 0 0 0 31 0 0 0 0 0 M - P Untreated' SPU45 0 0 0 0 0 0 0 0 0 0 0 0 L Pine E SPU99 13 0 0 0 0 0 0 0 0 0 0 0 S - MEAN 27 8 6 0 0 0 10 . 0 0 0 0 0

BRUT 38 0 0 0 0 0 0 0 0 0 0 0

8 Untreated BRU18 88 6 81 0 0 0 50 0 0 0 0 0 Birch D BRU38 100 13 0 0 0 0 50 0 0 0 0 0 A - Y MEAN 75 6 27 0 0 0 33 0 0 0 0 0

S SPU81 6 0 19 0 0 0 A 0 0 0 0 0 0 Mp Untreated SPU98 0 0 0 0 0 0 0 0 0 0 0 0 L Pine SPU125 E 81 0 0 0 0 0 0 0 0 0 0 0 S MEAN 58 0 6 0 0 0 0 0 0 0 0 0 16 DAY SAMPLES - PERCENTAGE FREQUENCY OF ISOLATION

3mm. Depth 10mm Depth 40mm Depth

ECOLOGICAL GROUP B PM S SR BS SM B PM S SR BS SM B PM S SR BS SM

BRU66 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Untreated BRU35 50 13 25 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Birch BRU90 100 0 50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

MEAN 50 4 25 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

BRT35 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Treated BRT66 100 19 0 0 0 0 0 0 0 0 0 0 • 0 0 0 0 0 0 Birch BRT90 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

MEAN 33 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SPU7 100 63 0 0 0 0 0 63 0 0 0 0 0 0 0 0 0 0

Untreated SPU21 75 100 13 0 0 0 0 0 50 0 0 0 0 0 0 0 0 0 Pine SPU79 50 63 88 0 0 0 25 25 31 0 0 0 0 0 0 0 0 0

MEAN 75 75 33 0 0 0 8 29 27 0 0 0 0 0 0 0 0 0

SPT7 100 13 0 0 0 0 38 0 0 0 0 0 0 0 0 0 0 0

SPT21 100 25 0 0 0 0 50 0 0 0 0 0 0 0 0 0 0 0 Treated Pine SPT16 100 25 38 0 0 0 100 0 0 0 0 0 0 0 0 0 0 0

MEAN 100 21 13 0 0 0 63 0 0 ~0 0 0 0 0 0 0 0 0 32 DAY SAMPLES - PERCENTAGE FREQUENCY OF ISOLATION

3mm Depth • 10mm Depth 40mm Depth

ECOLOGICAL GROUP B PM S SR BS SM B PM S SR BS SM B PM S SR BS SM

BRU 103 50 13 19 0 0 0 13 0 0 0 0 0 0 0 0 0 0 0

Untreated BRU60 50 44 38 0 0 0 50 0 0 0 0 0 0 0 0 0 0 0 Birch BRU13 69 0 88 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0

MEAN 56 19 48 0 0 0 23 0 0 0 0 0 0 0 0 0 0 0

BRT13 13 0 0 0. 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Treated BRT60 25 25 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Birch BRT 103 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0

MEAN 13 8 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SPU60 69 50 19 0 0 0 13 19 6 0 0 0 0 0 0 0 0 0

Untreated SPU97 50 13 81 73 0 0 6 0 50 13 0 0 0 0 0 0 0 0 Pine SPU127, 50 81 56 0 0 0 6 19 88 0 0 0 0 0 0 0 0 0

MEAN 56 48 52 4 0 0 8 13 48 4 0 0 0 0 0 0 0 0

SPT60 0 13 13 0 0 0 50 0 0 0 0 0 0 0 0 0 0 0

SPT97 50 50 13 13 0 0 44 6 6 0 0 0 0 0 0 0 0 0 Treated Pine SPT127 81 69 6 0 0 0 25 0 0 0 0 0 0 0 0 0 0 0

MEAN 44 44 10 4 0 0 40 2 2 -0' 0 0' 0 0 0 0 0 0 96 DAY SAMPLES — PERCENTAGE FREQUENCY OF ISOLATION

3mm Depth 10mm Depth 40mm Depth

ECOLOGICAL GROUP B PM S SR BS SM B PM S SR BS SM B PM S SR BS SM

BRU108 6 13 44 25 0 0 0 0 6 0 0 0 0 0 0 0 0 0

6 6 Untreated BRU93 6 31 33 0 0 50 13 0 100 0 0 0 0 0 0 0 Birch BRU73 100 100 0 33 13 0 13 13 19 0 0 0 0 0 0 0 0 0

MEAN 38 48 17 31 4 0 6 21 13 0 33 0 0 0 0 0 0 0

BRT73 100 44 13 71 0 0 13 13 0 0 0 0 0 0 0 0 0 0

Treated BRT93 0 19 0 8 0 0 6 0 0 0 0 0 0 0 0 0 0 0 Birch BRTIO8 13 0 0 25 0 0 0 0 0 0 0 0 0 0 0 0 0 0

MEAN 38 21 4 35 0 0 6 4 0 0 0 0 0 0 0 0 0 0

SPU41 50 100 6 25 0 0 31 31 38 0 0 0 0 0 0 0 0 0

Untreated SPU50 100 69 6 0 0 44 31 25 0 0 0 0 0. 0 0 0 0 0 Pine SPU100 100 25 6 21 0 0 25 0 6 0 0 0 0 0 0 0 0 0

MEAN 83 65 6 16 0 15 29 19 15 0 0 0 0 0 0 0 0 0

SPT41 50 25 0 83 0 0 56 6 0 38 0 0 0 0 0 0 0 0

SPT50 50 100 25 75 0 0 6 0 0 8 0 0 0 0 Treated 0 0 0 0 Pine SPT100 19 100 0 38 0 0 13 0 0 25 0 0 0 0 0 0 0 0

MEAN ' 40 75 8 65 0 0 25 2 0 -24 0 0 0 0 0 0 0 0 184 DAY SAMPLES - PERCENTAGE FREQUENCY OF ISOLATION

3mm Depth 10mm Depth 40mm Depth

ECOLOGICAL GROUP B PM S SR BS SM B PM S SR BS SM B PM S SR BS SM BRU88 19 25 88 33 25 0 31 25 19 29 50 0 0 44 19 0 88 0

Untreated BRU43 38 25 31 42 75 13 50 0 56 25 63 0 44 0 63 67 38 0 Birch BRU57 6 69 6 42 88 38 0 6 0 0 100 81. 13 38 6 25 0 25

MEAN 21 40 42 39 46 33 27 10 25 18 71 27 19 27 29 31 8 42

BRT43 0 6 0 21 0 44 0 56 6 17 0 0 6 13 0 71 0 0

Treated BRT57 0 0 0 59 0 0 0 7 0 88 0 0 0 0 0 0 0 0 Birch BRT88 0 19 0 88 0 0 6 6 13 4 0 0 13 13 0 0 0 0

MEAN 0 8 0 56 0 15 2 23 6 36 0 0 6 8 0 24 0 0

SPU44 100 100 38 17 75 0 100 31 63 8 0 100 19 0 0 0 0 0

Untreated SPU52 100 94 0 0 50 0 88 75 0 0 0 38 19 0 0 0 0 0 Pine SPU75 •81 100 0 0 38 0 75 69 31 25 0 0 0 0 0 0 0 0

MEAN 94 98 13 6 54 0 88 58 31 11 0 46 13 0 0 0 0 0

SPT44 50 100 0 92 0 0 100 6 0 4 0 0 0 0 0 0 0 0

SPT52 94 38 6 58 0 0 100 13 0 63 0 0 25 0 0 0 0 0 Treated Pine SPT75 81 88 0 38 0 0 100 0 6 13 0 0 0 0 0 0 0 0

MEAN 75 75 2 63 0 0 100 6 2 -26 0 0 8 0 0 0 0 0 265 DAY SAMPLE — PERCENTAGE FREQUENCY OF ISOLATION

3mm Depth 10mm Depth 40mm Depth

ECOLOGICAL GROUP B PM S SR BS SM B PM S SR BS SM B PM S SR BS SM

BRU95 0 25 25 8 100 0 0 25 19 0 88 0 0 31 13 0 100 0

Untreated BRU83 0 13 56 25 38 31 0 0 6 0 100 38 0 0 0 0 100 100 Birch BRU72 19 0 0 0 88 13 0 0 0 0 25 100 0 0 0 0 63 88

• MEAN 6 13 27 lI 50 40 0 8 8 0 71 46 0 10 4 0 88 63

BRT72 69 63 50 83 0 0 0 69 0 67 0 6 0 6 0 75 0 19

Treated BRT83 0 31 0 79 0 0 0 0 0 63 0 0 0 56 0 46 0 0 Birch BRT95 0 6 6 92 0 0 0 .0 0 42 0 0 0 0 0 29 0 0

MEAN 23 33 19 85 0 0 0 23 0 57 0 2 0 21 0 50 0 6

SPU12 19 44 0 33 25 0 44 38 0 21 75 0 38 50 0 0 25 0

Untreated SPU89 44 69 38 4 75 0 50 63 19 42 63 0 44 44 0 21 0 0 Pine SPU105 50 75 25 0 25 0 50 50 0 29 100 0 31 38 0 42 63 0

MEAN 38 63 21 13 42 0 48 50 6 31 79 0 38 44 0 21 29 0

SPT12 31 75 13 84 0 6 69 100 0 33 0 0 0 88 44 0 0 0

SPT89 69 50 0 100 0 0 50 75 6 96 0 0 38 63 0 0 0 0 Treated Pine SPT105 25 31 0 67 0 13 6 25 13 63 0 50 0 100 0 21 0 0

MEAN 42 52 4 83 0 6 42 67 6 -64 0 17 13 83 15 7 0 0 363 DAY SAMPLES — RELATIVE FREQUENCY OF ISOLATION

imm Depth 10mm Depth 40mm Depth

ECOLOGICAL GROUP B PM S SR BS SM B PM S SR BS SM B PM S SR BS SM

BRU123 0 6 25 0 50 50 0 0 63 0 75 6 0 6 63 0 75 13

Untreated BRU33 0 0 6 4 88 75 0 0 0 25 100 44 0 0 0 13 88 13 Birch BRU15 0 0 0 4 75 94 0 0 6 0 50 94 0 0 0 0 88 100

MEAN 0 2 10 3 71 73 0 0 23 8 75 48 0 2 21 4 83 42

BRT15 0 0 0 84 0 0 0 6 0 4 0 88 0 0 0 0 0 100

Treated BRT33 0 0 0 100 0 0 0 0 0 100 0 0 0 0 0 88 0 38 Birch BRT123 0 0 0 100 0 25 0 0 0 100 0 44 0 13 0 46 0 0

MEAN 0 0 0 94 0 8 0 2 0 68 0 44 0 4 0 44 0 46

SPU16 50 50 25 29 100 13 50 13 100 54 50 0 0 13 25 38 50 38

Untreated SPU48 50 31 0 4 100 31 50 50 13 25 50 0 0 63 0 0 25 0 Pine SPU51 50 13 50 4 100 50 50 13 50 0 75 13 25 0 0 0 25 0

MEAN 50 31 25 13 100 31 50 25 54 26 58 4 8 25 8 13 33 13

SPT16. 44 0 0 00 0 0 75 0 0 100 0 0 0 100 0 13 .0 . 0

SPT48 31 19 0 79 0 31 81 6 0 75 0 0 0 '00 0 33 Treated 0 0 Pine SPT51 75 0 0 96 0 0 25 25 0 83 0 0 0 50 0 38 0 0

MEAN 50 6 0 92 0 10 60 10 0 "86 0 0 0 83 0 28 0 0 214

APPENDIX 3: Computer Program, Sample Data, Sample Output

PROGRAM ROTTEN CIMPUT. OUTPUT. TES=P UT. TAPE6=OUTPUT) 0001R0T DIPEPSION IC(9)• ATITLE(75)• P0(20.9.7)• TTL(40.30)• STKCDC30) 0005R0T DIPENSION I51(30)• MRS(30). NSEC(30)• NOV (30) DIPE7ISION DK (30.260) . NFG (30) • IC0 (30) • T*F (20) COMMA 112.01(. 1-11 .STKCD. IST.CIRS.ICSEC•MOEP 00 996 I1 = 1. 1260 0020001 10 (11) = 0 0025R0T 996 CONTINUE 0030ROT DO 997 12 = 1. 6000 003590T OK(12) = 0.0 0040ROT 997 CONTINUE 0045ROT REPO (5.) I01. ( IC (I3) • 13= 1. ICT) DO 996 I5 = 1. IC(1) 0065ROT READ CS . 999) NT. ( TT!. (NT. I7) •17=1.30 ) 0070ROT 900 F0RM1T C 12. IX. 3011 ) ROT 906 CONTINUE 0080R0T READ (5.995) NGP. ( T1FG(14)•I4=1• PCP) 0056ROT 995 FORP7AT (4012 0060901 READ (S.1000) ( ATI7LE(I61.I8=1.75) 0085ROT 1000 FORMIT ( 7591) 0090901 IF C IC(6) .E0. 1 ) 1RITE01.1001) CATITLE(19) • I9=1.7S) 1001 FORMAT C //i• 1X. 75A1. 10X. 26H3C0TS PINE ANALYSIS SElaCTEO.//i/) IF c 1C C6) £0. 2 1 WRITE (6.1022) (ATITLE CI9) • 19=1.75) 1022 FOAM7AT C /ii. IX. 75A1. 10X. 23118I901 ANALYSIS SELECTED.AiA./2 DO 1002 N = 1. FIGP 0105R0T DO 1003 I11 = 1. 11FG M) 0110R0T READ (5.1004) STKCD 00 •I5T(11).NR5 CN) .NSEC (1).11OEP (N) .NP (I11). 2 (CPO(I11.K.L)• L=1.7). K$1.9) 0120ROT 1004 FORPAT ( A6. 13. 211. 212. 2X. 9(711) ) 0125ROT 1003 CONTINUE 0130ROT IF ( IC C2) .EO. 0 1 GO TO 1009 0135901 19117E ( 6.100S) N. STKCO CM) . 15700 . MRS Cro .IISEC CM *MEP CM) 1005 FORPi1T C IX. 5AHGROIW. IX. 72. SX•5PISTAKE. 2X. A6. SX. 1111ST491 2 1110SAMPLE TIPE. IX.I3.1X.4CDAYS.5X.51(STAKE.I2.5X.7HSECTI(N.12.SX. 3 %OEPTH. 13. 1X. 21419 0155ROT TR ITE 05.1005) 016CROT 1006 FORP7AT ( /.2X.3HFLO. IX. 2HNF. IX. 9(3X.1114T H 000 V 14)) DO 1007 112 = 1. MFG (N) 0170R0T MITE 05.1008) I12. AP (I12) • 100(I12.K.L) .1=1.7) .K=1.9) 1000 FORTAT OX. 12. 1X. I2.1X. 9(3X.2 (I1010 . 3I1. 2(1X.I1))) 1007 CONTINUE 0165ROT 1009 IF CIC(3) .LT. 1 1 GO TO 1040 9131 DO 1010 J = 1.11FG 00 0195ROT DO 1010 K =1.9 0200ROT • DK 01.1) = OK(I.1) + 1.0 - 020SROT IF C IC ON OT. 1 1 G0 TO 1014 C SCOTS PINE ANALYSIS C C TISSUE TYPES ROT CALL C3P1 ( N•J.K.1r1.5.2) '011090T IF ( POU.K.1) .10. 9 1 01(00.7) = DKC1.7) + 1.0 0215R0T IF CPO U.K.1) .19.9) G0 TO 1010 C HYPHAE ROT CALL 0391 C M. J. K. 2. 1. 3. 9 1 0220R0T IF C POU.K.2) .10. 0 ) OK131.8) = OKOM.S) + 1.0 0225ROT IF CPC (J•K.2) .EO.1.09.P0 (Jo K.2).E0.2.OR.(1(J.K.2).10.3) 231907 2 OK (N.12) _ OK (Mr 12) + 1.0 C ATTACK OR 0FGRAqA7I31 0235907 IT(PO(..1.K.3) •F0.0. AND •IOU•K.4) .EG.0.An0.PO ha( .5) .10.0) 2 01(01.13) _ OK (11.13) + 1.0 IF C PO (...1.1(.31 .GT. 0 .0R. PO (J.K.4) .GT. 0 .OR. PO U.K.5) .GT.0) 2 DK CN.28) = OK 01.26) + 1.0 CALL CDPP2 ( N. J. K. 2. 10. 14 ) C CELL HALLS ROT

IF'QO(...1.K.1).E0.1./40.POCJ.R.6).11.2)DR01.23) W3K011.23)+1.0 IF ( POU.K.1) .01. 1 .0M0. PO (J.K.6) .07. 2.1110. 19(J.K.6) ROT 2 .LT. S) DK 01124) = p(01.24) + 1.0 0245207 IF C POU.K.1) .10. 1.1110. )OU.K.6) .(E.5)0K071.23) =OK (M.25)+1.0 0230 IF C PO(J.K.7).GT. 0 .1113.10 (J.K.7).LT. 9)0K 01.26)=0K(1.261+1.0 IF( IOU.K.7) .02. 9 ) 01(01.27) = DK(11.27) +1.0 13260 C CA.CILATION OF VALUES FOR EACH TISSUE TYPE Il01VIOUALLY TALL 034PSP C M.J.K. 1.32) CALL 0317SP C M•J.K. 2.52) CALL CM PSP C M.J.K. 3.72) 00 TO 1010 C BIRCH ANALYSIS C C TISSUE TYPES 1014 CALL GI 1l(N.J.K.1.l(.21 CALL C3P1 ON.J.K. 1.2.2.4) CALL C39101.J.K.1.3.4.5) CALL CDPP10M.J•K.1.5.5.3) IF 0OU.K.1) .E0. 9 ) O(.01.7) = OK U1.7) + 1.0 IF CPC (J.K.1) .E0.9) G'0 TB 1010 C H1PH AE CALL COPP101.J.K.2.1.3.97) IFQ124J.K.2) .10.0) 0K01.5) = OKO1.8)+1.0 IF OM➢ U.K.2) .E0.1.OR.19 U.K .2) .C9.2.OR.PO U .K.23 .E0.3) 2 OK 01.123 = OK 01.1Z + 1.0 C ATTACK 139 OEORKIATION IF 0PO U.K.1) .10.4) GO TO 1012 IF CPO U. K.3) . E0.0. A10. PO (J.K.4) . E0.0. A10.10 0. K.5) .10.0) 2 01(01. 133 = DK (11.13) + 1.0 1012 IF C PO (.1.K.3) .CT. 0 0R. PO U.K.41 .GT. 0 .OR. POCJ.K•5) .GT.0) 2 OK 01.23) = 0K 01.23) + 1.0 CALL LOPP2 01.J•1(.2.10.20) C CELL HALLS IF CPO (J.K.1) .E0.3.111D.PO U. K.6) .GE. 1) OK U1.24) =OK 01.24) +1.0 IF000.K•1).EO.1.A10.P00•K.6) .1T.3)OK01.25) =OK (11.251+1.0 IFQO CJ•K.1) .E0.1.0M0.PO (J.K.6) .07.4)0K 01.25) 41K (11.25)+1.0 11 00 U.K.1).E0.2.fl10.10 U.K.7).GE.1)OK(1.26)=01((1.26)+1.0 IF OO (j. K.1) .10.5.1110. PO (J. K.7) .GE.1) OK 01.27) =000 01.27) +1.0 C TISSUE TYPES ISDIVIOUALLT CALL CO PS 01.J.K•18323 CALL CllP80I.J.K.2.52) CALL COIPSCN.J.H•3.72) CALL CLIPS 0I.J.K. 4.921 CALL CKl175131.J.K.5.112) 1010 CONTINUE 0295 IF C IC(8) .GT. 1) GO 78 1015 C SCOTS PM PERCENTAGES CALL PC (11.01((1.1) . 2• 28. 102) CALL PC (1. OK 01.2) • 32. 47. 132) CALL PC11.OK01.3). 32. 87. 152) CALL PC (1$. d( 04.4) . 72. 67. 172) 90 TO 1040 C BIRCH PERCFIOTAOES 1015 CALL PC (11.0K01.1) .2027.132) CALL PC (1.0K C11.2) .32.47.1621 CAL PC 171.13K (1.4) .52.87.182) CALL PC CN.0K 01.S).72.87.203) CALL PC 01.0K 01.6) .92.107.222) CALL PC CHICK (1.3) .112.127.2421 1040 IF C IC (4) LT. 1 GO TO 1002 IF (IC (6) .03. 1) CALL RESSP Cro SF( IC(6) .10. 2 ) CALL RES801) 1002 CONTINUE 0355 IF ( IC(S) .LT. 1) 03 TO 1029 215

IF C IC(6) .EO. I FRITE(6.1001) CATITLE(19). I9=1.75) IF C IC(6) .EO. 2 IRITE(6.1022) CATITLE(19). 19.1.75) 1029 READC5.) NGA. (ICtG(I20).120= 1 .MGA) 0375 IF C 1040(1).1T. 1) GO TO 1030 4AITEC6.1024) C IQCGC121). 121= I.NGA) 1024 FORMAT C /////.IX. 26NPOOLED RESULTS GROUPS. 3013) WRITE (6•7004) 7034 FORMAT (IX. /i/) DO 1026 129 = 1. MGA M = ICPGCI29) IRITE C 5.1005) M. ST)CC 0 CP0 . I51 O0 . PR5 00 .!SEC OO . P(DEP UO 1026 CONTINUE MC = SC (6) IF C NC GT. 1 ) GO TO 1011 CALL GiPAV C 1. MGA. IQ(G 102. 128. 2. PC) CALL C77PAV ( 2. MGA. ICMG 132. 142. 12. PNC) CALL ORPAV C 3. P01. IQ0 152. 162. 12. PC) CALL GRPAV C 4. MGA. 1QG 172. 182. 12. PC) GO TD 1029 1011 CALL GRPAV C 1. MGA. IOC 132. 157. 2. MC) (ALL GRPAV C 2. MCA. IQD 162. 177. B. PC) CALL GRO0V C 3. MGA. IMO 242. 257. 9. NC) CALL GRPAV C 4. MOO. 1CMO 182. 197. 6. PCC) CALL GRPAV ( 5. MGA. 1C1G 202. 217. 6. MC) CALL GRPAV C 6. MOO. 1QC 222. 237. 6. PC) GO TO 1029 1030 STOP 050010T END 0505ROT SUBROUTINE COPPSPCIN.JJ.KK.N1.P(2) DIMENSION M2(20.9.7). TR (40.30). OK(30.260) COMMON MG. OK. rn (ALL CPP3(N1.JJ.KK.M1.M2) (ALL CONPS(1ll.JJ.KK.M1.N2+1) CALL CI#'P4CNM.JJ.KK.M1.2.10.N2+2) CALL Crc6(NM.JJ.KK.M1.N2+11) CALL COPP7CPM.JJ.KX.M1.162+15) SCRAM END SUBROUTINE COMP5CNM.JJ.KK.11I012) DIMENSION M2(20.0•72. Tn c4D.3D) . OK (30.260) COMMON P0. OK. 711 CALL C)PP301I.JJ.KK.Ml.M2+4) CALL CCrP50CM.JJ.KK.M1.M2+5) CALL CrP6 CMM.JJ.KK.N1.N2) CALL COMP40M.JJ.KK.N1.2.10.N2+6) CALL CIlP7011.JJ.KK.M1.N2+15) RETURN END SUBROUTINE COP1 C MI.JJ.KK.LL. MM. N1. MDA) 0510ROT. DIMENSION 140(20.9.7) . 111.00.30). DK (30.260) C/14 M 10. DK. 111. PDK = MDA DO 2000 M = 111. MM 0525ROT IF C t0CJJ.KK.L L) .E9. M) OKCMI .MOK) = OK(M1 .NDK) + 1.0 053000T MDK = MDC + I 0535ROT 2000 CONTINUE ROT RETURN 054500T END 055000T SUBROUTINE COPP2 (M1. JJ. KI. MM. MM. MDA ) 0560ROT DIMENSION P13(20.0.7) . 711. (40.30) . DK (30.260) COMM ro. DK. TTL KK = K1 NCC = MCA 00 3000 MI = MM. MCM 0575R1T

M = Ml - 1 0560R11T IF C MDR .E0. 19 W TO 3003 IFOOCJJ.KK.5) .E0. M) GB 18 3001 IFOOCJJ.KK.4) .E0. M) G8 TO 3001 - IF OO CJJ. KK. 3) .E0. M) W TO 3001 00 TO 3002 3003 IF C M]w.KK.5) .GT. 5.) GO TO 3001 IF C POCJJ.KK.4) .GT. S) GO TO 3031 IF C 10 CJJ.KK.3) .GT. 5 GO TO 3001 W TO 3002 3001 OK01101DK) = OK 011.11K) + 1.0 3002 MDK=MDK+1 3000 COMTIPI. E 0600R1T RETURN 0505881' EMD 0610ROT SUBROUTINE COMM 011. JJ.KK.NT.PDK) 0615ROT DIMENSION 113(20.0.7). 1711.(4°.3°). DK C30.260) Carport PO. DK. 111. ROT IFOOCJJ.KK.1) ME. MT ) G6 TO 2501 IF ( POCJJ.KK.2) .EO. 1) W TO 2500 IF C MOCIJ.KK.2) .E9. 2 ) 00 TO 2500 IF C 1CJJ.KK.2) .E9. 3 ) W T8 2500 GO TO 2501 2500 DK 911.1053 = OKCPII.MOK) + 1.0 2501 REPLI 064005E END 06.95867 SUBROUTINE CO'P4 011.JI.KI.PT.MM.Ml1.NDA) 0650R6T DIMENSION 10(20.9.7). 111-C40.303. OK (30.260) 93110N 112. DK. TTL ROT JJ■Jl KK = KI PCK • MDA IF C POCJJ.KK.1) PE. MT 2 RETURN DO 4000 MI - 111.Mf1 0665RBT M = M1 - I 0670ROT IF C NCDK .E9. MCA + 5 ) W TB 4003 AFC MUW.KK.5) .EO. PD GB TO 4001 SF( IO(JJ.KK.4) .E9. M) 00 TO 4001 ITC MOOJJ.KK.3) .W. PD DO TO 4001 00 TO 4002 4003 IF C MOCJJ.KK.5) .GT. 5 ) W TO 4001 IF C POCJJ.KK.4) .GT. 5 ) W TIP 4001 IF ( POCJJ.K1C.3) .OT. 5 2 W TO 4031 W TO 4002 4001 05 011.l05) = OK 011.1010 + 1.0 4002 10K = PICK + 1 0655401' 4000 CONTINUE 060010T RETURN 0695R0T ETV 0700ROT 'SUBROUTINE COPPS C MI. JJ. KK. PMN. MCA ) DI7121510M M0(20,9.7), TTL (40.30)C OR(3ON260) carton M2. DK. rn IF C Pow.KK.1),IE. PM) W T3 0000 IF C Pow.KK.5) .PNE. 0 2 W TO 0000 IF C NOCJJ.KK.4) .PE. 0 2 0a TO 6000 IF ( M2CJJ.KK.3) ..ME. 0 2 W TO 5000 OK ( 161. PDA) = DK 011.1DA) + 1.0 8000 RETURN END SUBROUTINE COMPS CN2.JJJ. KKK.NT. MDA) DIPEMSIOM POC20.9.7) . T1LC40.30) . OK (30.260) COMMON M2. DK. Tn ROT PCK=M0A $ MI = M2 R JJ=JJJ $ KK=555 IF 012(JJ.KK.1).ME.PIT) 03 TO 5000 216

IF CPO w.KK •2).E0.0)DK (NI .)OK)=0K(Ml.11010+1.3 IF CFO (JJ.KK .2).E0.1)DK(N1.N0K+1) =OK (N1.110K+1)+1.0 IF (P0 (JJ.KK .2).E0.2)0K(NI.PO(+2)=0K(NI.N0S+2)+1.0 IF via (.1.1.KK.2).EO.3) DK (NI .110/(+3) =OK (211 • NOK+3) +1.0 5000 RETURN END SAAROUTOE 031117 011• JJ. KK 'MN .NOA) DIPENSIOIN P0 (20.9.7) • TTL (40.30) . OK (30.2601 031.11311 10. DK. TTL Hdc = PDA IF C10 (.0 .KK .1).PE.ho GO TO 7000 IF ( PO(JJ.KK.3) .GT. 0 .HR. 2 PO(JJ.KK.4) .O1.. 0 .OR. PO(JJ.KK.5) .07. 0 ) 3 OK MOCK) . OK (111.110K) + 1.0 7000 RETURN DO SUBROUTINE PC C NI. 5V. 11• IF. WA) 0710R6T DIPENS11101 10 C20.9.7) • TR. (40.30) • DK (30.250) COMM 0. DK. TTL ROT IF C DV .LT. 0.99 ) GO TO 5001 DP = 100.0 / 6V 0725651 (OK = PDA 00 5000 I = U. IF 0700R0T OK 011.10K) • DR (1/1.1) . 6P ( OK = P0K + 1 0740ROT 6000 CONTINUE 0743ROT RETURN 07SOR0T 8001 MK = PER D0 6002 1 II.IF 0 141.NDt) = 0.0 110K= POK+1- 6002 CONTINUE RETURN END 0755R5T SUBROUTINE RESSP •( Ni) DIPENSION ma (20.9.7) . rR <40.30) • OK (30.260) DIPE1SION 151(30) • MRS (312) MSEC(30)• MEP (30).STKCO(30) COMM PO.OK.rm. STKCO .15T. NM. MSEC. NOEP N • Ni 0775R0T warm( 6.100S) M. STKCO OD . 1ST(10 .PIRS til .MSEC CM) .NOCP CN) . 1005 FORMT ( ///. IX. 5NGROUP• IX. 12. 5X.5I+STRKE. 2X. A6. 5X. 2 11fl5APPLE TIPE. 1)613. SX.4WAY5.5X.5L(STPKE.I2.5X.7MSECTIDN•I2•5X. 3 5LGEpTR. 13. IX. 244t0 IRITEG6.1013) 0330 • 1013 FOFFNT c /i . 13X. 51{7111E. 16X. 131•119ICR00UAORATS. 8X. 2 DHTRALLEID• OX. 14NRAY PARD10411 . OX. 1271RAY TRAC)EID. //. 0340 3 33X. 4 (14 4 110. PERCENT. 5X)) 0345 WRITE (6•7000) 4TTL(I•115).115.1.30)• OK (II .1) 0760ROT 7000 FORMAT( IX. 30111. F6.1) 0755401 00 7001 516 = 2. 7 117 . 115 + 100 079056T TRITE (5.7002) (TR (116. I15) .115.1.50) • OK O(.116) .OK 01417) 7002 FORFPIT ( IX. 30A1. P6.1. 2X. F13.4) 060540T 7001 CONTINUE 0510R6T DO 7010 120 • 6.11 1aITEt6.7O04) (TR (120.115) .115=1.30100K 01.120) .OK QI. I2o.l00) . 2OK (11.120+35).O(01.120+135) *DR (N.I20+55).0KCM.120+155) . 30K 01.120465)•0k CM .120+155) 7010 CONTINUE 00 7003 118 • 12. 22 0615401 WRITE 15.7004) C TR (I16.115)•115.1.30). DK 01.118)• OK(N.I16+t00). 0020607 2 OK (Ms 116+20)• Df(11.I15+120)• OC(N.I18+ 0) . 3 DK el 110+140) .0K Qf.118+60) • DK 01.110+1601 7004 FORMAT C IX. 30111 • 4 M6.1.21(.313. 4.313 ) 0035ROT

7003 CONTINUE 0840ROT I18 = 26 MITE (6.7004) ( TR (116.115) .115=1.30)• 0K 01.116)• OK 01.115+100)• 0620ROT 2 OK 01.116+20)• OK eh 118+120) 0K 01.118+40) . 3 OK 01.118+140) .01(01.115460)• 01( 01.116+160) 00 7005 119 = 23. 27 0645607 IBiTF(5.70O2) (TTL(I19.115).115.1.303.01( 04.I19) .OK 0(.119+100) 0135ROT 7005 Carainue 0660R0T TRITE (6.7009) 7009 FS1TR71T 42X. /// ) RETURN DO SUBROUTINE RDA (111) DSMEJ4S1014 10(20.9.7). TTL(10.30). OKC30.260) 011015I0,1 IST (30) • MRS (30) • MSEC (30) • MOEP (30) .31KC0 (30) COPTEN 10.OK.TR.STKCD. 1ST. MRS. NSEC. PREP D Ni TRITE( 6.1005) N. 51XCO00• IST MOOS CPO .M5EC CIO .NOEP 00 . 1005 l03211T ( ///. IX. 540fAOlA. SX. I2. O)6OMS7AKEr 2X. A6. 5X. 2 1111151:'PLE TIME. IX. U3.1XA30AT5.5X.5MSTWCE.12.5X•711SEGT10M.12.5X. 3 SPEEPT14. I7. lx. SURD MDT (5.21313) 2013 FORTNT C /i.13X.514TITLE 16x.13Tf1101BOUADRATS• 6x.6IFIBRE5. 2 7X.161FIDRE+PAR EICIN 1 O. 5X.4R11Y5• 9X.15MVESSEL INC TALL. 3 2X.15100E35EL EST TALL.//.33X.6(141 M0. pI:CFNT.3X)) lRITE(6.7000) (TTZ (1.1151.115.1.30) DK 01.1) 7000 FOAPAT( IX. 30111. P8.1) 00 7001 115 • 2. 7 117. 115 + 130 RRITE06.70021(T1L(I16.115)•I15.1.30). D(01.1153.DK 01.117) 7002 rearm. C IX. 30111. F5.1. 2X. 18.4) 7001 CONTINUE D0 7010 120.6 .23 WRITE 48.7012) (TTL(120.115).115=1.30) .0K Ole I20).OK01.120+130) . 2 DK (1.1201•24) .01( 01.120+154) ..)K 011120+104) .01( (11.120+234) 3 .OK 01.120+44) .OS 01.120+174) .0K (11.720+64) . 4 DK 01.120+194) .OK 01.120484) . DK 01.120+214) 7012 FOJhWr C IX. 3061. 6CF6.1.2X.F8.4.170) 7010 =VIM( 00 7015 119 • 24.27 1017E (5. mom (TTL (119.115).115.1.30).0K01•119).OK OW 119+130) 7015 CONTINUE MITE (6.7009) 7009 FORPAT (2X. /// ) WREN DC) SUBROUTINE 951PAV C NV. NOA6. IC1rt1G. II. IF. NTS. NCA D110451OP1 10120.9.7). TO. (40.30). DK (30.260) .ICN03(30).4(30) COMM TO. 01( • TTL ROT NOAH • MGM TRITE ( 5.6002) c T'TL CMV. II) . 11. 1.30) 8002 FORPAT ( /ii . IX. 3061 ) 1RITE46.6004) (TR (40.11).11•1.30) 8004 FORPAT (IX. 30111) WRITE (6.1a23) 0405 1023 FORPAT ( /• 13X. 514TITLE. 19X. 41FE1W. 4X. 9(PLUS SERB. 2X. 0410 2 10)611115 SIŌ1. 3X. 931STAND EAR. 3X. 9HSTAM0 DEV. 3X. BOAR 1,110E. 3 4X. SCOUT VAR. 3X. 2PDF. A 0425ROT 10 • MTI 0885ROT 00 8000 POK • 11. IT 086080T 1X1 6001 127 • 1. PCGAA 0890R0T N • IC130(137) 0895ROT 11(527) • OK (11.101(1 0900ROT 0001 CONTINUE tn)CnaT 217

CALL AVRGCA. NGAA. N3) 0915R0T 113 = 113 + 1 8000 CONTINUE 0925R0T IF (IIV.LT.2) RETURN IF C 1CZ .GT. 1) RETURN N3=8 DO 8005 I28=IF+1.IFa5 DO 8006 I29=1.NGAA N=ICNGGCI29) 11(129)=0R(11.828) 8006 CONTINUE CALL AVRG (R.14GAA.143) N3 = N3+1 8005 CONTINUE REMAIN 0990ROT END 0935ROT SU6ROUTINE AVRG C X. KIM. 142) 0940ROT DIMENSION 10(20.9.7). TT).(40.30). DI(30.250). X130) COItUi N0. DR. 111 ROT N = NOF =0 SM= S50= MAR= XPLUS= XMINUS= SERR= SOEV= VAR= COEFF= 0.0 QO 1712 I=1.NT75 SIFSI+XCI) SSQ=SSO + XCI) I X(I) N=N+1 1712 CONTINUE IF C 5M .GT. -1.0E-10 .ANO. S11 .LT. 1.0E-10) GO TO 1714 AN=N MAR = 51'VAN IF ( N .EO. 1 ) GO TO 1714. NOF = N-1 IF C ASS CSSO-51'R7mAR) . LT.1.0E-5 ) GO TO 1714 VAR = (550 - SM I MAR) / C AN - 1.0) SOEV = SORT (VAR) COEFF = (SOEV / MAR)* 100.0 SERR = 50EV / SORT (RI) XPLUS = MAR + SERR XMINUS = MAR - SERR 1714 WRITE(8118000) ITT).(112.I30)1430=1.30).2 AR.XPLUS.XMINUS.SERR.SDEV. 2 VAR.COEFF. NOF ROT 8000 FORMAT C IX. 30A1. 7CF10.3.2)3 . I4) ROT • RETURN ENO 218

Sample Block of Data as Collected

0 21111 DATA FOR 591 DAY SAMPLES — 3M1 DEPTH — BIRCH UNTREATED BRU028591110301 130253013025301302530130293013029301324530530254053245405324540 BRU028591110302 210030323003042200303130003013004301300430130004013004401300440 BRU028591110303 900000090000009000000110072011000201100420130073013027301100830 BRU028591110304 100094012009401000940100093010009301000930110093011009301100930 BRU028591110305 900000020003032200304130093090000009000000110093020003032000303 BRU028591110306 200030320003041100040200030320003041000040110005011000501100050 BRU028591110307 110093011009301100930110093011009301100930900000090000009000000 BRU028591110308 130594013059401305940130594013059401305940400000040000004000000 BRU028591110309 132583013258301325830132894013289401328940534794053479405347940 BRU028591110310 410000040000004000000110794011079401107940130794011079401107940 BRU028591110311 115794011579401157940110794011579401157940115794011579401157940 BRU028591130301 300001030000103000010130043013005301300430110043051004305100441 BRU028591130302 110093011009301100930110093011009301000030200002420000132000024 BRU028591130303 130592011009201305930510494053059415357941110793011079301107930 BRU028591130304 110094011009401300940110793011079301107930510094151079411107940 BRU028591130305 130044013004401300440110043013004301300430510043053004305100430 BRU028591130306 110473011004301107930110043011004301100430110043011047301104730 BRU028591130307 300001030000103000010400000040000004000000110044011004401100440 BRU028591130308 110793011009301100030110093011009301100930110793011009301100930 BRU028591130309 110003011007301100030110593011059301105930200002320000241100030 BRU028591130310 110793013009301105930110093011009301100930530044151009415305930 BRU028591130311 200002420000142000024200001320000132000013200001420000242000013

Each line represents the data from 1 quadrat (9 microquadrats). The code for that quadrat is followed by the data from each of the 9 microquadrats, seven numerals per microquadrat. The code shows the stake number BrU028 (untreated birch 28), the sample time (591 days) the replicate stake sample number (1 [ of 5 ] ) , the section number (1 [ of 2 or 3 ] ) , the depth (03mm) , the quadrat (1-10 or 11).

219

Sample Output

Data for each section printed first, followed by analysis of data. Results for replicate sections then pooled and analysed.

DATA FtA 591 DAY SAMPLES - 3MN DEPTH - BIRCH UNTREATED BIRCH MMILYSIS SELECTED

GROUP I STAKE DRUM SMPLE TIME 501 DAYS STAKE I SECTION 1 DEPTH 3 MN

TITLE MICi00UADRATS FIBRES F/BRE+PAREPC HVr71 RAYS VESSEL PC WALL .ESSEL EXC IALL M8. PERCENT NO. PERCENT MS. PERCENT N1. PERCENT NO. MERG=1HT NO. PER[E111. MICIOOUAORATS 90.0 FIBRES 67.0 67.6768 FIBRES + PAROCTom 6.0 6.0606 RAY 11.0 11.1111 VESSEL (INCL. HALL 0 0 VESSf1 KLUMEN BMLY) 6.0 6.0606 OUTSIDE Q.RSSIFICATIOM 9.0 9.0909 - HTPHAE ABSENT 18.0 18.1818 6.0 8.9552 0 7. 63.6364 5. 83.3333 HYALINE HYPHAE 34.0 34.3434 32.0 47.7612 0 1. 9.0909 1. 16.666 PIGPIDDED HYPHAE 3.0 3.0303 1.0 1.4925 0 2. 16.1818 HYALINE IMD PIGMENTED WPM( 35.0 75.3535 26.0 41.7910 6. 100.0000 1. 9.0909 wmP:AE PRESENT 72.0 72.7273 61.061.0 91.044691.0446 6. 100.0000 4. 36.3636 1. 16.666 M ATTACK 8.0 8.0808 8.0 11.9403 0 6. 100.0000 PIT PENETRATION 0 0 0 0 0 WALL PENETRATION 16.0 16.1616 13.0 19.4030 3. 50.0000 LOSS OF 62REFRIMI'ETCE 11.0 11.1111 0 0 0 11. 100.0000 WILL mum (TYPE ~ewe 11.0 11.1111 6.0 8.95528.9552 5. 83.333383.3333 CLASSICAK. SOFT ROT CAVITIES 24.0 24.2424 21.0 31.3433 3. 50.0000 8R51DIMmTCETE ATTACK 53.0 53.5354 50.0 74.6269 3. 50.0000 BONEHOLE'S 21.0 21.2121 16.0 26.6657 3. 50.0000 PIT ENLARGEMENT 7.0 7.0707 7.0 10.4475 0 WALL TRIMMING - 6ASIDIOIMETE 46.0 46.4646 43.0 64.179164.1791 3. 50.0000 SOME FORM OF ATTACK 75.0 75.7677 59.0 88.059788.0597 6. 100.0000 11. 100.0000 PORE THAN 1 VESSEL IALL 0 0 MOT 3 MOR 4 FIBRE WALLS 6.0 6.0606 HORIZONTAL WILLS - RAYS 11.0 11.1111 HORIZONTAL WILLS - rreacipmeol 0 0

GROUP 2 STAKE BRUO2O SAMPLE TIME 591 DAYS STAKE 1 SECTION 3 DEPTH 3 M1'1

FLDMF TH000V TH000 VH TM000 VH TM000 VM TH000 VH TH000 VH TM000 VM TM000 VM THDODvH 1 1 3 0 000 1 3 0 000 1 0 3 0 000 1 0 1 3 004 3 0 1 3 005 3 0 1 3 004 3 0 1 1 004 3 0 5 1 004 3 0 5 1 004 4 1 2 2 110093 1 100930 1 100930 1 100930 1 100930 1000030 2000024 2000013 2000024 3 3 1 3 059 2 1 1 009 2 0 1 3 059 3 0 5 1 049 4 0 5 3 059 4 1 5 3 579 4 1 1 1 079 3 0 1 1 079 3 0 1 1 079 3 0 4 4 1 1 009 4 1 1 000 4 0 1 3 009 4 0 11 079 3 0 1 1 079 3 0 1 1 079 3 5 1 009 4 1 5 1 079 4 1 1 1 079 4 0 5 5 130044 1 300440 1 300440 1 1 00430 1300430 1 3 004 3 5100430 5300430 S 1 30430 6 6 1 1 047 3 1 1 004 3 0 1 1 079 3 0 1 1 004 3 0 1 1 004 3 0 1 1 004 3 1 1 004 3 0 1 1 047 3 0 1 1 047 3 0 7 7 300001 300001 0 3 00001 0 4 000000 4000000 400000 1 100440 11 00440 1 100440 8 8 1 1 079 3 1 1 009 3 0 1 1 000 3 0 1 1 009 3 0 1 1 009 3 0 1 1 000 3 1 1 079 3 0 1 1 009 3 0 1 1 009 3 0 9 9 1 10003 1 1 00730 1 1 00030 1 1 05930 1 105930 1 1 0503 2000023 2000024 11 00030 1010 1 1 0793 1300930 1 1 05930 1 1 00930 1 1 00930 1 1 0093 5300441 5 00941 5305030 1111 200002 2000014 2000024 200001 3 20000 1 3 200001 20000 14 200002 4 20000 13

GROUP 2 STAKE 6RU028 SAMPLE TIME 591 DAYS STAKE I SECTION 3 DEPTH 3 MT

TITLE MICiOOIJAORATS Flames FIBAE4PAgEOHYP RAYS VESSEL INC WALL VESSEL EXC WALL M8. PERCENT M6. PERCENT M8. PERCENT MO. PERCENT Ne. PERCENT N8. PERCENT HDAADRRYS 99. FIBRES 63. 63.6364 FIafsS + PRn71C4WA 13. 13.1313 RAY 14. 14.1414 VcSSEI. (INCL. WALL 6. 6.0606 VESSEL KLAREN ELY) 3. 3.0303 OUTSIDE CLASSIFICATION 0 HYPHAE ABSENT 24. 24.2424 1. 1.5673 0 14. 100.0000 6. 100.0000 3. 100.0000 HYALINE HYPHAE 58. 56.5659 50. 79.3651 8. 61.5385 P/GmEN ED MYPAMAE O 0 0 HYALITE AND PIGMENTED HYPHAE 17. 17.1717 12. 19.0476 S. 38.4615 WPM( ARMADA' 75. 75.7576 62. 98.4127 13. 100.0000 MO ATTAQ 25. 25.2325 5. 7.9365 0 14. 100.0000 6. 100.0000 3. 100.0000 PIT PEETRATIOM O 0 0 FALL PENETRATION O 0 0 LOSS 6F BIREFRINGENCE O 0 0 FALL DECAY (TYPE UNKNOWN) 27. 27.2727 20. 31.7460 7. 53.6462 CLASSICAL SBFT ROT CAVITIES 10. 10.1010 7. 11.1111 3. 23.0769 BIŌIONANCETE ATTACK 47. 47.4747 40. 63.4921 7. 53.8462 BOREHBLES 17. 17.1717 15. 23.8096 2. 15.3846 PIT ELAIā'E?ENT O 0 0 FALL THINNING - BASIDIOILETE 43. 43.4343 36. 57.1420 7. 53.6162 D 0 SOME FOON OF ATTACK 71. 71.7172 56. 92.0635 13. 100.0000 0 0 TORE THAN 1 VESSEL WALL 6. 6.0606 MOT 3 NOR 4 FIBRE WALLS 2. 2.0202 HORIZONTAL WALLS - RAYS 14. 14.1414 HORIZONTAL WALLS - FIORE/PARER 7. 7.0707 220

DATA FOR 591 DAY SAMPLES - 3MN DEPTH - BIRCH Un7R£ATED 6IRCH ANALYSIS SELECTED

POOLED RESULTS GROUPS 1 2

GROUP 1 STAKE 641X326 SAMPLE TIME 591 DAYS STAKE 1 5CKTIB4 1 DEPTH 3 MI GROUP 2 STAKE 6RU028. SAMPLE TIME 591 DAYS STAKE 1 SECTION 3 DEPTH 3 MN

MICR001M10RATS

TITLE MEAN PLUS SETS! MINUS SERB 5TAND ERR STAMP DCV ugataMCe C71Err Mw OF FIBRES 65.657 67.677 63.636 2.020 2.657 6.162 4.351 1 FIEmS 4 PRRENCYPN 9.596 13.131 6.061 3.535 5.000 24.997 52.103 1 RAY 12.626 14.141 11.111 1.515 2.143 4.591 16.971 1 vESSEL (INCL. I..L) 3.030 6.061 .000 3.030 4.265 18.365 141.421 1 vESSEL 4UPEN OILY) 4.545 6.061 3.030 1.515 2.143 4.591 47.140 1 OUTSIDE t.as5dCATI5n 4.545 9.091 0 4.545 6.426 41.322 141.121 1 WT9lEAM 66501T 21.212 24.212 16.182 3.030 4.265 16.365 20.203 1 HYALINE HYPHAE 46.465 56.566 34.343 12.121 17.142 293.646 36.893 1 PIGMENTED WAWA( 1.515 3.030 .000 1.515 2.143 4.591 141.421 1 HYALITE AND PIGMENTED HYPHAE 26.263 35.354 17.172 9.091 12.656 165.269 46.954 1 HYPHAE PRESENT 74.242 75.756 72.727 1.515 2.143 4.591 2.666 1 68 ATTACK 16.667 25.253 6.061 6.566 12.142 147.434 72.653 1 PIT PENETRATION 0 0 0 0 0 0 0 0 MALL PENETRATION 6.061 16.162 0 6.001 11.426 130.599 141.421 1 LOSS or 6SREFR111G;1ACE 5.556 11.111 0 5.556 7.857 62.726 141.421 1 wMLL DECAY (TYPE 1H 61.0 19.192 27.273 11.111 6.061 11.426 130.599 59.546 1 CLASSICAL SOFT ROT CAVITIES 17.172 24.242 10.101 7.071 9.999 99.990 56.232 1 BASIDISFACETE ATTACK 50.505 53.535 47.475 3.030 4.265 18.365 8.465 1 66REH6LES 19.192 21.212 17.172 2.020 2.657 8.162 14.666 1 PIT ElAlARG=TEMT • 3.535 7.071 -.000 3.535 5.000 24.997 - 141.421 1 WALL TWINNING - 6A51OI0rfYCETE 44.949 46.465 43.434 1.515 2.143 4.591 4.767 1 SOME FORM OF ATTACK 74.242 76.766 71.717 2.525 3.571 12.754 4.810 1 MORE THAN 1 vESSEL WALL 3.030 6.061 .000 3.030. 4.265 18.365 141.421 1 !OT 3 IBR 4 FIBRE WALLS 4.040 6.061 2.020 2.020 2.857 8.162 70.711 1 HORIZONTAL WALLS - RAYS 12.626 14.141 11.111 1.515 2.143 4.501 16.971 1 NORIZBAITAL WALLS - FIDP>E,PAREH 3.535 7.071 -.000 3.535 5.000 24.997 141.421 1

FIBRES

TITLE MEAN PLUS SEAR MINUS SEAR STAND ERR STAND DEV vARIANNCE COEFF V66 OF HYPHAE ABSENT 5.271 6.953 1.567 3.664 5.210 27.143 96.636 1 HYALITE HYPHAE 63.563 79.365 47.761 15.602 22.347 490.403 35.156 1 PIGMENTED HYPHAE .746 1.403 .000 .746 1.055 1.114 141.421 1 HYALINE AND PIGMENTED HYPHAE 30.419 41.791 19.046 11.372 16.0622 256.632 52.666 1 HYPHAE PRESENT 94.729 96.413 91.045 3.664 5.210 27. Y7 5.500 1 65 ATTACK 9.936 11.940 7.937 2.002 2.631 8.015 26.467 1 PIT PETE'T66TIDN 0 0 0 0 0 0 0 0 TALL PENETRATION 9.701 19.471 0 9.701 13.720 165.236 141.421 1 Less OF erIETRt7[ENcE o 0 0 0 0 0 0 0 WALL DECAY (TYPE 1103660 20.351 31.746 6.955 11.305 16.116 259.710 79.180 1 CLASSICAL sari' ROT CAVITIES 21.227 31.343 11.111 10.116 14.306 204.670 67.396 1 BASIDl9fl'OTE ATTACK 69.059 74.627 63.492 5.567 7.673 61.992 11.401 1

6ORE766LE5 25.336 26.666 23.810 1.526 2.161 4.670 6.529 1 PIT E7LAROE63fT 5.224 10.446 0 5.224 7.365 54.576 141.121 1 WALL THINNING - easIDI6MYLETL 60.661 64.179 57.143 3.518 4.975 24.754 6.202 1 same FORM BF ATTACK 90.062 92.063 65.060 2.002 2.631 8.015 3.144 1

rI6WE5 t PARE11;41rn

TITLE MEAN PLUS SETRPR MINUS SCAR STAND ERR STAG DEv vMRIANCL COAT VAR Or HYPHAE ABSENT 0 0 0 0 0 0 0 0 HYALINE PYPNiAE 30.766 61.536 .000 30.756 43.514 1603.491 141.421 1 PIGMENTED HYPHAE 0 0 0 0 0 0 0 0 HYALINE AND PIG ENTER HYPHAE 69.231 100.000 38.462 30.769 43.514 1663.491 62.654 1 HYPHE - PRESENT 100.000 0 0 0 0 0 0 1 NO ATTACK 0 0 0 0 0 0 0 0 PIT P[lETTRRTI9N 0 0 0 0 0 0 0 0 WALL PAE?ETAATI431 25.000 50.000 -.000 25.000 35.355 1250.000 141.121 1 LOSS OF BIREFRINGENCE 0 0 0 0 0 0 0 0 WALL DECAY (T1PC CHKN640 66.590 03.333 53.646 14.744 20.851 434.747 30.398 1 CLASSICAL SOFT ROT CAVITIES 36.536 50.000 23.077 13.462 19.037 362.426 52.103 1 62tOI6M7LETE AT1AAC7K 51.923 53.646 50.000 1.923 2.720 7.306 5.230 1 6e(R6I46LE5 32.692 50.000 15.365 17.306 24.477 590.112 74.670 1 PIT DLRRPEYEN7 0 0 0 0 0 0 0 0 WALL TNI111In3 - e2tOIGfl2ETS 51.923 53.646 50.000 1.823 2.720 7.366 5.238 1 SOME FORA 65 ATTACK 100.000 0 0 0 0 0 0 1

TRAY

TITLE MEAN PLUS SER R MINUS SEIRRR STAR ERR STAND DEV VARIANCE COE7FF MAR OF WYPHAE ASSENT 81.816 100.000 63.636 18.162 25.713 661.157 31.427 1 HYALINE HYPHAE 4.545 9.0a1 4.545 6.426 41.322 141.121 1 PIGMENTED HYPHAE 9.091 18.162 9.091 12.656 165.269 141.421 1 HYALINE AND PIP2'ENTED HYPHAE 4.545 9.001 4.545 6.126 41.322 141.421 1 WRAC PRESENT 18.182 36.364 16.162 25.713 661.13 141.421 1 Na ATTACK 50.000 100.000 50.000 70.711 5000.000 141.421 1 PIT PENETRATION 0 0 0 WALL PENETRATION 0 0 0 LOSS OF SIRElRINGEME 50.000 100.000 50.000 70.711 5000.000 141.421 1 WALL DECAY (TYPE UMO6+0 CLASSICAL SOFT ROT CAVITIES 6ASIDIOflTE7E ATTACK BOREHOLE5 PIT ENLARGEMENT &LL THINNING - eA5IDIOMNYCErE SOME FORM OF ATTACK 50.000 100.000 50.000 70.711 5000.000 141.421 1 22]

\o£SSEL (UCI.. ~L) _ fAICE CIlfJT _ TIn.E I'£AIt PLUS SDIIl MlPIJ5 SDIIl SlAIC) OIR 5TA1O DEV D'"

~ A8SfJIT 50.000 100.000 -.000 50.000 70.711 !5OOO.ooo l~l.

H'I'PI« ~T 0 0 0 0 0 0 0 0 re ATTRO(. 50.000 100.000 -.000 50.000 70.711 !5OOO.000 1"'1.<421 1 PIT P!l"£TRATlOtl 0 0 0 0 0 0 0 0 ~L P!l"£TRATIOtI 0 0 0 0 0 0 0 0 LI!SS Of" IURETR INCiEI1CE 0 0 0 0 0 0 0 0 ~L OECIIY (T'rPE l}I'1I(.NIlW'1) 0 0 0 0 0 J 0 0 Cl ASS ItAL StFT IIIlT CAV ITlES 0 0 0 0 0 0 0 0 er ·.IOI~ ATTI'IO(. 0 0 0 0 0 0 0 0 t JQDIClLES 0 0 0 0 0 0 0 0 HT £l'lLRRG£I£tIT 0 0 0 0 0 0 0 0 ~L ~INNIIIG - IIASIOI!lI'fI'CETE 0 0 0 0 0 0 0 '0 SOt£ f1lRt1 Of" ATTACIt 0 0 0 0 0 0 0 0

\o£SSEL CLUIO 0tIl. Y)

_ fAICE CO£I'T _ TIn.E t£AtI PLUS SEAR MINUS S£RR STAIIO ERR sTAioto DEV D'" H'I'PI« AlSSEI1T ell. 1587 100.000 113.333 11.333 11.7M l:l1!._ 12.l1'5li 1 II~ 11'1£ II'I'PI« 8.333 111.1587 0 11.333 11.7M 1:l1!.IIBII. 1"'1.~1 1 PI~ 11'11'1« 0 0 0 0 0 0 0 0 H~!1'£ AIC) P IGl'£l'tTEO II'/PIIA£ 0 0 0 0 0 '0 0 0 ~~T 11.333 111.1587 0 11.333 11.7M l:l1!._ 1... 1.~1 1 I'IIJA~ 100.000 0 0 0 0 0 0 1 PIT PDCTRfITIIlI1 0 0 0 0 0 0 0 0 ~L ~TIIlN 0 0 0 0 0 0 0 0 LItSS CII'" llIRETRIIIGEIU 0 0 0 0 0 0 0 0 ~L DECAy (T'rPE l}I'1I(.NOW'f) 0 0 0 0 0 0 0 0 Q.ASSItAL StFT IIIlT CAVITIES 0 0 0 0 0 0 0 0 8IIS IOI!lI'fI'CETE ATTl'ICX. 0 0 0 0 0 0 0 0 eoAEHIlLES 0 0 0 0 0 0 0 0 PIT £I'lL ARGEI"1EI'ff 0 0 0 0 0 0 0 0 ~L ~INNIIIG - IlASIDItlI'1'I'tZTE 0 0 0 0 0 0 0 0 SOt£ f1lRt1 CII'" ATTI'IO(. 0 0 0 0 0 0 0 0 222

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