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PENCIL STREAK, STAIN AND DECAY IN EUCALYPTUS AND SYNCARPIA IN NEW SOUTH WALES

AUTHOR

D. W. Edwards

Forestry Commission of New South Wales Sydney 1982

1

Forestry Commission of N.S.W., Wood Technology and Forest Research Division Post Office Box 100, Beecroft, 2119 New South Wales, Australia

Published 1982

National Library of Australia Card Number ISSN 0729-5340 ISBN 0 7240 6668 3

Printed in Australia by Star Printery Pty. Ltd. 21 Coulson St., Erskineville, N.S.W. 2043

2

CONTENTS

Page ABSTRACT 4

INTRODUCTION 5

PENCIL STREAK IN EUCALYPTUS 7 OBLIQUA

Macroscopic features Microscopic features Distribution Nature of pencil streaked wood Fungi in pencil streaked wood Production of pencil streak in vitro Role of insects

DECAYS ASSOCIATED WITH PENCIL 14 STREAK AND TREE INJURIES OF E. OBLIQUA

Macroscopic features Microscopic features Fungi causing decay

PENCIL STREAK, STAIN AND DECAY IN 16 E. PILULARIS

Initial investigation Whole cross section study Materials and methods Results Discussion Conclusions

PENCIL STREAK, STAIN AND DECAY IN 18 E. NITENS

Materials and methods Results Discussion Conclusions

DARK HEART IN TURPENTINE 19

ACKNOWLEDGEMENTS 20

REFERENCES 21

APPENDICES 24 1 Forester's classification of Eucalyptus obliqua

test logs cut at"Banshea State Forest 2 Culture media 3 Cultural and other characteristics of basidio­

mycetes isolated from Eucalyptus spp.

3

ABSTRACT

PENCIL STREAK, STAIN AND DECAY IN EUCALYPTUS AND SYNCARPIA IN NEW SOUTH WALES

D. W. Edwards

The condition of pale-coloured eucalypts known as pencil streak is a reaction between the living tree and wound invasion by pioneer fungi introduced by ambrosia beetles. The nature, development and significance of pencil streak is discussed and the condition shown to be an unreliable indication of loss of wood strength.

The relationship of decay to pencil streak in living eucalypts and the nature of stains in eucalypt logs are also discussed.

Some other work on the stains and decays of eucalypts and turpentine is reported.

4

1. INTRODUCTION Occasional problems concerning the presence of

stain and decay in sawn wood of native species have been brought to the attention of the Forestry Commission of New South Wales over the last 20 years. Many of these defects have been accepted as normal for the species concerned and most work on them has not concerned itself with amelioration of the problem in living trees .

Pencil streak i an old term fir t applied to thin black lines of stain injarrah (Tamblyn 1937; Rothberg 1937). This report deals primarily with pencil streak in Eucalyptus ob/iqua L. Herit. (messmate), E. pilularis Srn. (black butt) and E. nitens Maiden (shining gum), but other investigations by Commission officers are also reviewed.

Figure 1. Eucalyptus obJiqua mining timbers showing (a) pencil streak, (b) brown stain , and (c) an insect gallery. (LC 388)

Figure 2. Eucalyptus obliqua mining timbers showing (a) brown stain , and (b) decay pockets . (LC 388)

5

.------------------------------------------------------------------------------- ---

Figure 3. Pencil streak in Eucalyptus obliqua heartwood. Insect galleries occur at (a) and pencil streak at (b) . (LC8713)

6

b

PENCIL STREAK, STAIN AND DECAY IN EUCALYPTUS

AND SYNCARPIA IN NEW SOUTH WALES

D.W. Edwards Research Paper No. 1. 1982

ERRATA

Caption on page 7 is deficient in data. The appended amending slip may be superimposed.

4a,b. Eucalyptus obliqua l. Herit. (messmate)

4c. E. viminalis Labill (manna gum)

4d,e. E. fastigata Deane et Maiden (brownbarrel)

4f,g. E. sieberi l. Johnson (silvertop ash)

4h,i. E. microcorys F. Muell. (tallow wood)

The Commission apologises for the oversight.

2. PENCIL STREAK IN E UCA LY PTUS in timber u ed either to support cantilevered platforms at the ore face, or as prop and shoring. The platforms, which must support six men and heavy drilling machinery up to 7 m above the tunnel floor, are exposed to blast and are constantly wet. Their collapse would almost certainly cause serious injury or death . Props and shoring have less critical criteria but freedom from incipient decay is mandatory.

OBLlQ UA

In 1964, trial shipments of messmate were sent from Oberon to Broken Hill , New South Wales, for tests below ground in the silver-lead mines . Because of mine safety requirements any loss of strength due to hidden causes was considered to be completely unacceptable

a b

I

Figure 4. Pencil streak and stain in wood of five Eucalyptus spp. (Insect galleries may be seen in Figures 4c, 4e, 4g, 4h, 4i.) (Fe 3384)

4a,b. Eucalyptus obliqua L. Herit. (messmate)

E. viminalis Labill. (manna gum)

E. fastigata Deane et Maiden (brown barrel)

4f,9. E. sieberi L. Johnson (silvertop ash)

4h ,i. E. microcorys F. Muell. (tallow wood)

7

The quality of messmate available from Oberon in 1964 was not good, pencil streak, decay and brown stain being commonly found alone or together. The mine management was concerned with problems of safety, and the prominent appearance of pencil streak and its apparent association with decay led them to use it as an indicator of unsound wood (Edwards 1964).

A field inspection of mess mate logs from Banshea S. F . was made by a group of forestry , sawmilling and mining officers in 1964 (Appendix I) . It revealed a high incidence of decay, and defective timber, which was reflected in the quality of mining timbers cut from similar material from the same forest (Figs . I and 2). Material of this type was rejected at the pit top.

2.1 Macroscopic features In transverse log sections of messmate from Banshea

S . F. pencil streak followed the direction of the rays. Thus, if a transverse section was cut in exactly the right horizontal plane, long, thin black streaks were seen to extend from pith to outer heartwood . Pencil streak has not been detected in sapwood (Fig. 3).

On the tangential face of boards from pencil streaked logs the defect occurs as lenticular flecks of varying length, depending upon the number of ray cells intercepted by the saw-cut.

In the eucalypts listed in Table I the appearance of pencil streak is the same except for jarrah, a dark red timber, in which the black markings are larger and more diffuse. However, Tamblyn (1937) gives a description of pencilled jarrah very similar to that for E. obliqua. Figs. 4 and 5 show the appearance of pencil streak in a number of species.

Similarities between pencil streak and black stains around insect galleries in mess mate suggests that the two may have a common origin.

2.2 Microscopic features Examination of pencil streaked wood under the

microscope showed that the dark flecks were due to coloured deposits in the vessels and surrounding ray parenchyma cells (Fig. 6). Some of this material had penetrated into the surrounding fibre lumina by way of the pits. It is formed originally in the ray parenchyma, and in the early stage of deposition appears as a hemispherical granular mass with a pit opening at its apex.

These deposits were very similar to heartwood crystals described by Chattaway (1952) but were seldom found to be associated with fungal hyphae. No evidence of pencil-streaked wood was found more than a few fibre cells from rays , but individual fibres were occasionally filled with similar dark material in an apparently random fashion . The deposition of these substances follows the normal path of water movement in hardwood .

Amorphous , brown , non-birefringent deposits were found in large amounts in vessels , whether tyloses were present or not. When tyloses occurred , deposits were frequent in the spaces between them . They were found also in ray parenchyma and fibres and it was a combination of the two types of deposits which produced pencil streak .

Abundant polyphenol-like substances also occurred in ray parenchyma and fibres. This material was

8

transparent and did not appear granular even in older heartwood . It does not appear to be a cause of pencil streak.

Within vessels close to insect tunnels aggregations of thick-walled, dark-brown septate hyphae were found. These hyphae were irregular in shape, anastomosis was frequent , and in some specimens clamp connections, which indicate decay fungi , were present. However there may have been several fungal species present including decays unrelated to pencil streak.

A feature of most pencil-streaked zones was the rarity - or absence of fungal or bacterial attack, determined either by the presence of fungal hyphae or bacterial cells , or by the breakdown pattern of cell walls.

2.3 Distribution Examination of 738 authentic wood samples of

eucalypts in the Forestry Commission of New South Wales collection, supplemented by several thousand wood samples used for teaching and field collections which were made during this study showed pencil streak to be present in 23 eucalypt species (Table I).

From these results it appears that pencil streak, occurs chiefly in the subgenus Monocalytus but some affected species are scattered throughout Symphomyrtus.

Table I. Occurrence of pencil streak< in Eucalyptus Spp.1

Subgenus Species

Monocalyptus E. marginata Donn ex Srn. E. jacksonii Maiden E. muellerana Howitt E. laevopinea R. T. Bak. E. baxteri (Benth.) Maiden and Blakely E. eugenioides Sieb. ex Spreng. E. cameronii Blakely and McKie E. oblonga DC E. pilularis Srn. E. obliqua L'Herit. E. delegatensis R. T. Bak. E. jastigata Deane and Maiden E. sieberi L. 10hnson E. radiata Sieb. ex DC. E. piperita Srn. E. campanulata R. T. Baker and H . G. Smith

Symphomyrtus E. guilfoylei Maiden E. ery thronema Turcs. E. camaldulensis Dehnh. E. viminalis Labil!. E. badjensisde Beuz. and A. Welch E. microcorys F. Muel!.

Uncertain E. scabra Dum-Cors.

t Euca lypts classified in order suggested by Pryor and Johnson ( 1971 )

The occurrence of pencil streak in E. guilfoylei, E. jacksonii and E. marginata, all Western Australian species and its absence from plant groups other than eucalypts suggests that the plant responses which give rise to this feature are of great antiquity.

kino which is a dark-coloured phenolic exudate found in veins or in pockets in which the parenchyma bridges have broken (Hillis and Brown 1978). Sub lances which appear similar but are not kino occur in E. obliqua. It is most likely that they are produced by ray parenchyma cell at the sapwood j heartwood transition zone following stimulation by fungi present in the sapwood.

2.4 Nature of pencil streaked wood Tamblyn (1937) described substances associated

with pencil streak in the red timber E. marginata as

a

e •

,

.-

t •

Figure 5. Pencil streak and stain in wood of seven Eucalyptus spp. (Insect galleries may be seen in Figs. 5b and Se.) (FC 3387)

Sa. Eucalyptus laevopinea R. T. Bak. (si lvertop stringbark)

5b. E. baxteri (Benth,) Maiden et Blakely (brown stringybark)

5c. E. marginata Donn. ex Sm. (jarrah)

5d. E. pilularis Sm. (blackbutt)

Se. E. punctata DC (grey gum)

Sf. E. siderophloia Benth. (grey ironbark)

5g. E. fastigata Deane et Maiden (brownbarrel)

9

a

b c

d e

Figure 6. Deposits in cells of Eucalyptus obliqua L. herit. associated with pencil streak. (Polaroid - D. W. Edwards) a. Fungal hyphae in vessel affected by pencil streak, b-e. dark deposits in vessels, ray parenchyma and fibres.

10

COOH COOH

Sugars - HoO-OH OO-OH OH OH

Shikimic acid pool

o 5-dehydro shikimic acid

\ COO·R COOH

ellagitannins ~ HO OH ~ ~ HOVOH -~ HOVOH

OH OH

ellagic acid gallic acid esters gallic acid

Figure 7. A poss ible biosynthetic pathway for the formation of ellagic acid . (After Birch 1963; Brown 1966; Geissman 1963a, 1963b; Hathway 1957; Hathway and Seakins 1957, Humphries 1965; Towers 1976.)

The introduction into wood of additional oxygen following insect injury or fire may be a necessary condition for this transformation which might also be due to metabolic changes brought about by drought or disease.

Gardner and Hillis (1962) reported large amounts of ellagic acid in eucalypts (Table 2) and said that the amount of free ellagic acid varies both between and within species. The outer heartwood contains more ellagic acid than inner heartwood, especially in large trees and some occurs in sapwood (Hemingway and Hillis 1971).

Table 2. Occurrence of ellagic acid in Eucalyptus obliqua L. Herit. (After Gardner and Hillis 1962.)

Sample

Inner heartwood Outer heartwood Inner heartwood Outer heartwood Sapwood

Age of tree (yrs)

220

180

Trunk Ellagic acid Diameter (% oven-dry

(mm) wood)

508 1.15 2.85

406 0.50 1.85 0.10

Tannic acid and tannins (either condensed or hydrolysable) are present in eucalypts and both gallotannins and ellagitannins may be present in the same eucalypt species (Hillis 1953, Hillis and Carle 1959, H umphries 1965). Hillis and Carle say that many pale-<:oloured eucalypts contain large amounts of ellagitannins, most of which can be removed from fresh wood by prolonged extraction with water and alcohol. Polyphenols of red-<:oloured eucalypts are largely insoluble in these solvents.

With the exception of E. moluccana Roxb. , the recorded distribution of ellagic acid in eucalypts is confined to the Macrantherae and Renantherae. Whether this is significant in terms of phylogeny is not known. It is possible that ellagic acid may have a much

11

wider distribution among eucalypts than present knowledge indicates, and Bate-Smith (1963), who noted that it has not been found in ferns, gymnosperms or monocotyledons, thought that it had ''the possibilities of being a remarkably accurate taxonomic guide".

Clusters of crystals thought to be ellagic acid were found in the heartwood of many eucalypts in association with fungal hyphae (Chattaway 1952). For these eucalypts which contain large amounts of ellagitannins or ellagic acid , she suggested that, as the water content of the heartwood decreases during heartwood formation, the resultant super-saturated solution of ellagic acid could be "nucleated" by the hyphae to form crystals. In this study little evidence of fungal hyphae associated with ellagic acid could be found, and this may be explained by the observation of Hillis (1953) that ellagic acid is present in all eucalypt tissues even when fungal hyphae have not been detected, suggesting that ellagic acid is a normal metabolite.

Biosynthetic pathways for ellagic acid have been discussed (Geissmann 1963a, 1963b; Hathway 1957; Hathway and Seakins 1957; Neish 1964; Brown 1966) and their ideas are summarised in Figure 7. Brown warned that our knowledge of these steps is not satisfactory but agrees that the formation of gallic acid from glucose via the shikimic acid path is likely (Brown 1966). Gallic acid - sugar esters could then be formed but the esterification reactions are unknown . Finally Hathway (1957) and Hathway and Seakins (1957); have shown that mushroom polyphenoloxidases convert gallic acid to ellagic acid quantitatively. A possible pathway for the formation ' of extra ellagic acid and ellagitannins in which fungi play a role therefore exists in living trees.

Other possible causes of stains around beetle galleries have been suggested (Gadd & Loos 1947). Verrall (1943) attributed similar stains to ambrosia fungi intensified by insect secretions or other organisms but Gadd & Loos think it more likely to be due to the strain of ambrosia fungus carried by the vector beetles.

2.5 Fungi in pencil streaked wood

(a) Materials and Method One hundred and eighty wood chips each I mm long

of E. obliqua containing pencil streak were inoculated on to malt / yeast extract agar and incubated at 27°C for 12 weeks.

(b) Results Fungi isolated are shown in Table 3.

Table 3. Fungi isolated from pencil streaked areas of Eucalyptus obliqua L. Herit.

Number of Species Isolates

Basidiomycetes (decays) - Poria mollusca (Fr.) Cooke 2 - Unidentified species 2

Pioneer and / or staining fungi - Cephalosporium sp. I - Geosmithia swiftii (Swift) PiU' I - Leptographium sp. 3 - Paecilomyces varioti Bainer 2 - Penicillium spp. 6 - Phialophora Sp.2 6 - Unidentified yeasts 9

Saprophytes and contaminants - Monilia sp. I - Mucor spp. 2 - Pestalotia sp. I

I Formerly Penicillium bacillisporum Swift. 2 Probably P. jasligiala Lager. Lund. and Melin.

The basidiomycetes including Poria mollusca will be discussed later. Whilst they were found adjacent to pencil streak zones they occurred also in brown areas from the pith outwards. This appears to be a classical example of the succession of pioneer and decay fungi (Shigo 1979) as components of the decay processes which follow injuries to living trees .

The role of Geosmithia swiftii is unknown but it is commonly found as a pioneer fungus of wounded trees. The genera Phialophora and Leptographium contain a number of fungi which cause dark stains in timber, and species of the latter are frequently imperfect stages of Ceratocystis which cause blue stains of both eucalypts and pines.

Yeasts are common components of wound-altered wood and as contaminants which grow rapidly on freshly cut, wet timber.

2.6 Production of pencil streak in vitro To determine whether pencil streak could be

produced under laboratory conditions the following test was made:

(a) Materials and method Blocks of E. obliqua, 30 mm3, with a moisture

content of approximately 40% were cut from non­stained areas of a pencil streaked log. On the tangential face of each block four holes 25 mm deep by 2 mm

12

diameter were made. The blocks were sterilised by fumigation with propylene oxide vapour and placed in 220 g glass jars on sterile washed vermiculite , wetted to capacity.

After 2 weeks incubation to stabilise moisture content each hole was inoculated with one of the following test fungi:

Geosmithia swiftii Leptographium sp. Paecilomyces varioti Phialophora (Prob. P. Jastigiata) (two isolates) Poria mollusca Yeast (unidentified)

There were six blocks for each isolate and one group of 6 controls. The inoculated blocks were incubated for 12 weeks at 27° C and 70% relative humidity in glass tanks. At the end of the test the blocks were split at the holes and examined for evidence of pencil streak.

(b) Results With the exception of two Phialophora spp. isolates,

growth was good. In Phialophora inoculated blocks growth was either sparse or absent. No evidence of pencil streak was found in any of the blocks.

These results suggests that pencil streak formation requires the existence of living host cells.

2.7 Role of insects Association of pencil streak with insect damage

seems likely in view of the constant presence of platypodid (ambrosia) beetles such as Austroplatypus incompertus (Schedl) Browne and associated black sta ins. This species was originally named Platypus incompertus by Schedl (1968), but subsequently F. R . Browne (1971) erected a new genus, Austroplatypus, to which he assigned P. incompertus. The type specimens are in the Victorian National Museum. This species is one of the few platypodids associated exclusively with living, apparently healthy trees (Browne 1971) and it attacks a number of N.S.W. eucalypts (Table 4).

There is an abundant literature on ambrosia beetles and their associated fungi: Baker 1963; Bakshi 1950; Batra 1963a, 1963b, 1967; Batra and Francke­Grosmann 1961 ; Batra and Michie 1963; Cooke 1977a, 1977b; Davidson 1955; Farris 1963; Farris and Funk 1965; Faulds 1973; Fernando 1960; Finnigan 1963; Francke-Grosmann 1956a, 1956b, 1957, 1963, 1965, 1967; Gadd and Loos 1947; Jones 1959; Lagerburg, Lundberg and Melin 1927; Leach 1940; Litchwark 1978; Norris and Baker 1967; Schneider-Orelli 1911, 1913; Verralll943; Webb 1945; Wilson 1959). Between them a composite picture may be built up of fungi associated with such stains. There is considerable confusion as to the identity of many of the organisms, but Batra (1967) has done much to clarify this situation.

Ambrosia beetles are widely distributed in Australia attacking sta.nding fire-killed trees (Hogan 1948) freshly felled logs, and green, sawn timber. They do not attack seasoned wood. Black halos surrounding insect tunnels are a feature of platypodid beetle attack, and result from introduction of fungi by adult females . These fungi , which may be specific to a particular insect species, line the tunnel walls and provide food for developing larvae . (Cooke 1977a, 1977b).

Figure 8. Mycetangia of Austroplatypus incompertus (Schedl) Browne. a. Mycetangia on pronotum of female. b. Mycetangial orifices. (Photos R. Johnson).

13

The mechanism by which fungal spores are transferred from tree to tree was once thought to be accidental, e.g. deposition of spores on insect hairs. Batra (1963a), Farris (1963) Francke-Grosmann (1963), and others showed however that many ambrosia beetles have special organs - mycetangia ­in which spores and hyphal fragments are kept viable but inactive until needed. Mycetangia may be located on the pronotum, or behind the coxa below the sides of the pronotum. The mycetangia of A. incompertus are heart-shaped, and located on the upper surface of the pronotum (Figure 8).

A. incompertus (usually male adults) tunnel horizontally through bark into inner heartwood.' Secondary tunnels are then bored, often at right angles to the entrance tunnel, i.e. in a tangential direction. Above and below the main galleries brood chambers are then bored. The entrance hole is probably the exit hole.

Colony viability requires maintenance of an unobstructed gallery to the outside air' . To do this the insect must move to the surface at frequent intervals to eject frass , and a short tube of chewed up wood is often built out from the bark.

In E. ob/iqua from Banshea SF a long, rambling type of gallery has been observed (Fig. 3). The galleries are circular to oval in cross-section and lined by a jet black layer of fungal hyphae . Dark deposits similar to those in pencil streaked areas extend about 2 mm into the wood. The brood chambers have a similar lining.

The length of various stages of this insect's life cycle is not known. The beetles are strong flyers and their distribution has interesting parallels with that known for pencil streak as can be seen by comparing (Tables I and 4).

Table 4. Eucalyptus spp. attacked by Austroplatypus incompertus (Schedl) Browne.

Subgenus Species Authority

Monocalyptus E. muellerana Howitt I E. laevopinea R. T. Bak. I E. macrorhyncha F. Muel!. ex Benth. E. baxteri (Benth.) Maiden and Blakely E. pilularis Srn.

2 2 1,4 1,2,4 2,4 1,2,4 3

E. obliqua L'Herit. E. delegatensis R. T. Bak. E. Jastigata Deane and Maiden E. consideniana Maiden

Symphomyrtus Uncertain

E. sieberi L. 10hnson E. dives Schau. E. botryoides Srn. E. scabra Dum-Cours.

1,2 3 3 2

I. Campbell , 2. Harris, 3. Harris (probable) (Campbell, K. Forestry Commission of New South Wales - Personal communication), 4. Browne (1971).

3. DECAYS ASSOCIA TED WITH PENCIL STREAK AND TREE INJURIES OF E. OBLlQUA 3.1 Macroscopic Features

Decay in pencil streaked wood is best seen in freshly sawn logs as a water-soaked irregular heartwood zone which may extend almost to the sapwood. It appears to enter the tree around knots, and this is clearly shown during conversion. Affected wood differs in colour from normal heartwood , being pale to very dark brown or sometimes bright yellow. In extreme cases softening occurs, and there may be severe decay near the pith. In freshly awn timber such damage is prominent but is more difficult to see as the timber dries out.

I Campbell , K. Forestry Co mmission of ew South Wales -Persona l communicat io n.

14

This is a typical example of the decay process (Shigo 1979) where initial invasion by pioneer fungi is followed by decolourization and finally decay.

3.2 Microscopic Features Hyphae associated with these decays are thin ,

hyaline and with or without clamp connections. Irregularly shaped chlamydospores occasionally occurred in the vessels . There are no other distinguishing microscopic features except that where severe yellow staining occurs brown thick-walled hyphae with infrequent clamps are present in both vessels and rays .

J

3.3 Fungi causing Decay Identification of decays in logs and sawn timber is

seldom possible unles sporophores are present. Despite efforts by Waiters and Da Costa there is as yet insufficient information on Australian wood-rotting fungi to allow satisfactory identifications on the basis of cultural characteristics as is done in Canada (Nobles 1965). This situation is likely to persist for some years.

(a) Iso/ation of Decay Fungi Discs 150 mm thick were cut across full trunk cross­

sections of selected logs of either E. obliqua or E. pilu/aris. Other pieces 150 mm )( 50 mm2 were taken from brown-stained areas of several flitches of the same two species. All samples were wrapped immediately in clean polythene sheet and isolated the following day.

From each piece a number of small samples (50 mm )( 250 mm2) were cut , depending on the size of the disc, but never less than three. Each sample was flame sterilized on all faces, split with a sterile chisel and from the centre of each, three chips were transferred on to each of three petri dishes containing malt / yeast extract/ glucose agar (Appendix 2).

The fungi isolated from E. obliqua which had basidiomycete features are shown in Table 3. Isolates FC 859, 860 and 911 from E. pi/u/aris ex Manning River National Forest also showed typical decay features.

(b) Cultural characteristics of decays isolated from E. obliqua and E. pilu/aris

N obles (1965) in her definitive work on cultural characteristics of wood-inhabiting Hymenomycetes gives a mUltiple choice key for identification of 149 Canadian species of wood decays based on published data on their cultural characteristics. Although it is probable that some wood decays of E. obliqua and

other eucalypts are uniquely Australian, others are cosmopolitan. For this reason, and despite the lack of published data on the cultural characteristics of Australian species it was decided to record the Australian isolates in terms of Noble's key, with variations described by Fritz (1923); Davidson et a/ (1938); Etheridge (1957), and Jorgenson and Vejlby (1953). Isolates FC 859, 860 and 911 from E, pi/u/aris failed to survive in culture and isolates FC 2841,3047 and 3417 were substituted for them.

Cultures were grown on malt / yeast extract agar as described by Nobles except that "Oxoid" agar and malt extract were substituted for "Difco-Bacto" products.

All microscopic examinations were made on material mounted in 5% aqueous potassium hydroxide and stained with 1% aqueous phloxine. Noble's gum guaic solution was used to test for formation of extracellular oxidases.

Results are shown in Appendix 3.

(c) Sporophore production Etter (1928), Koch (1962), Niederpreum (1963),

Plunkett (1953), and Raper and Krongelb (1959) studied the role of environment and nutrition in the formation of wood decay fruiting bodies. Despite these investigations successful laboratory production of sporophores of wood decays remains an art rather than a science. The technique which has proved most successful to date is that of Badcock (1941, 1943) as modified by Tamblyn and da Costa (1958). This method was further modified for this study. Other changes suggested by Yurchenko and Warren (1961) were considered and rejected as being too specialised, or likely to be useful with a very limited range of species.

Figure 9. Modified 8adcock sporophore test. (LH 19225)

15

A basa l medium of sawdust plus nutrients was prepared (Appendix 2). The medium was wetted until it just failed to absorb further water, then a small amount of dry medium was added to absorb any free water. It was then loosely packed into 300 mL bottles until level with the top of the neck and the bottles capped with aluminium foil and autoclaved at 103 kPa for 20 minutes.

When cool the sterile media was inoculated with 5 mm cubes of the test fungus taken from 10-20 days old malt / yeast extract Petri dish cultures, and incubated at 27°C until the mycelia had penetrated throughout the containers.

From clear sapwood of Pinus radiata D. Don, 45 mm x 50 mm2 blocks were cut so as to expose two transverse faces at right angles to the short axis of the tree . The blocks were routered to give a 45 mm diameter hole, 19 mm deep, which allowed the block to be fitted tightly over the neck of the culture bottle . A similar hole 5 mm deep was made in the top of the block to act as a water reservoir.

When the fungus had grown sufficiently the aluminium foil caps were replaced under aseptic conditions by the wooden blocks which had been previously autoclaved at 103 kPa for 20 minutes , and the block and bottle neck covered with sterilized polythene sheet. The assembly was incubated at 27° C until hyphae appeared at the edges of the blocks . The assembly was then transferred to glass tanks , the water well in the top of each block filled with sterile water , and the tank covered with sterile polythene sheet. The assembly was incubated at room temperature in subdued light until sporophores appeared. (Figure 9.) Occasional replenishment of the water reservoir was necessary.

Of the six wood decays tested only two produced sporophores. One of these was identified in 1967 by Walters l as Poria mullusca (Fr.) Cooke, who was of the opinion that it agreed in all details with Cunningham's description except for the rot type. Waiters however , does not consider that Cunningham's rot types are always correct. The identity of the other was not established.

(d) Decay potential of isolates from E. obliqua and E. pilularis

The ability of a fungus to decay timber in service is not always the same as in living trees , Duncan (1958), Feist et al (1971) , Hartley (1958). Many heart rots do not persist after the tree is felled and the wood must be transformed by pioneer fungi before serious damage occurs.

Seven basidiomycetes, four from E. obliqua and three from E. pilularis were compared with two common wood-decaying fungi supplied by CSIRO Division of Building Research.

The fungi used were: (a) Poria mollusca (Pers. ex Fr.) Cooke. FC 511.

Source: E. obliqua-Banshea State Forest , Oberon, N.S.W. This identification is tentative but Cunningham (1965) records it from both Europe and New Zealand and Lloyd (1910) from Australia. It produces a brown rot.

'N. C. WaIters Division of Forest Products CS IRO. Personal Communication.

16

(b) Unidentified Basidiomycetes. Source: Isolates FC 518 , FC 519, and FC 705 were from E. obliqua in Banshea State Forest, Oberon, and Isolates FC 859, FC 860 and FC 911 from E. pilularis, Manning River National Forest , Taree, New South Wales.

(c) Coniophora sp. (probably c. olivaceae (Fr. ex Pers.) Karst.). Source: Division of Building Research, CSIRO. Cartwright and Findlay (1958) describe the species as a brown cubical rot of heartwoods, which grows well in warm climates and is common in central and northern Europe and America. Cunningham (1965) says it occurs in Australia , New Guinea and New Zealand .

Screwcap jars (110 g) with a wide mouth were filled with 5 g of vermiculite , and 30 mL of distilled water. On this wetted bed of vermiculite were placed 20 cm2

feeder strips of flame kurrajong veneer 2 mm thick and the assembly autoclaved at 103 kPa for 20 mins with the caps loosely screwed in place. The feeder strips were then inoculated with the test organism, the metal caps screwed down but not so as to exclude all air. The assembly was then incubated for 14 days at 27°C.

Test blocks were cut from inner heartwood of E. obliqua from Oberon, N.s.W. and air dried to give a final weight of about 4 g. All were selected to give a similar density group and cut to present a machined tangential face in contact with the test organism. The test blocks were dried at 105° C for 24 hours to determine oven dry weight , then soaked in tap water for 10 days to give a moisture content of about 80%. The blocks were then autoclaved at 103 kPa for 30 mins.

After the test fungus had grown sufficiently on the feeder strips (14 days) a test block was placed on each strip. The lids were again partially screwed down and the jars incubated in glass tanks over a free water surface at 27°C for either 9 or 10 weeks.

Results - See Table 5.

All isolates tested on wood block substrates produced some decay after 9-10 weeks but no significant differences were obvious between them. The low level of decay (4-6%) suggests the need for caution when interpreting the results.

4. PENCIL STREAK, STAIN AND DECAY IN E. PlLULARIS

4.1 Initial investigation At the request of the Commission's Engineering

Aspects Research Group, fifty-seven samples of E. pilularis were examined for bacteria, fungi and other symptoms of wood biodeterioration (Edwards 1979a). These samples were from complete log cross-sections taken from butt and top logs of six trees supplied by that Group.

Hand cut sections in transverse, radial longitudinal and tangential longitudinal directions were made from each sample and mounted in lactophenol aniline blue. All samples were examined for deposits within the cells, tyloses, fungi and bacteria.

Table 5. Relative decay potential of six basidiomycetes grown on Eucalyptus obliqua heartwood at 27° C for 9 and 10 weeks.

Growth Number of Average Isolate Origin time replicates weight loss s.d.

(weeks) (%)

Poria Sp.1 E. ob/iqua 10 10 4 1.65 FC 518

" 9 6 4 1.26

FC 705 "

9 6 5 2.59 FC 859 E. pilularis 10 8 5 1.64 FC 860

" 10 10 6 3.00

FC911 "

10 10 4 1.32 Control

" 10 8 )2 1.39

I Probably P. mollusca. 1 Probably due to contaminants.

Cell deposits were found in most sections examined and were readily identifiable in all three planes. Similar deposits had been previously described by Dadswell (1972), who distinguished them from kino-like substances. There was no apparent relationship between these deposits and variations in the mechanical strength of the samples.

The occurrence of ellagic acid deposits similar to those described by Chattaway (1952) was very variable and did not appear to be associated with fungal hyphae. However, as Chattaway notes, ellagic acid is deposited only in cells which are already dead and from which hyphae may long since have disappeared by autolysis. Ellagic acid is very common in pale­coloured eucalypts and in some species is responsible for problems during conversion to paper pulp.

Fungal hyphae were common in sapwood and more particularly in heartwood. In nearly every case the hyphae were thin and hyaline and without clamp connections. Since no fruiting bodies were produced it was not possible to determine whether these hyphae were of pioneer fungi or wood decays. No deterioration of cell walls either from the lumen inwards, or within the cell wall itself was found in any

samples, which is presumptive evidence that they were not wood decays. In a few samples dark coloured hyphae of a type commonly associated with ambrosia beetles were noted. No fruiting bodies were produced so this could not be confirmed.

Well developed tyloses were present in most samples and in the genus Eucalyptus this is a reliable indication of heartwood. In many cases the interfaces of the tyloses were covered with extraneous materials.

4.2 Whole cross section study Samples from a further 5 trees of regrowth black butt

supplied by the Engineering Aspects Group were next examined to determine whether wood destroying fungi and / or bacteria were associated with changes in mechanical properties at two levels in each tree. The history of these samples and their mechanical and physical properties have been reported elsewhere (Ghali et al 1979).

4.3 Materials and Methods Complete cross-sections were cut from butt and top

logs of five trees as shown in Figure 10.

interior dark coloured zone exterior exterior

2 3 4 5 6

Figure 10. Diagram of a typical discoloured sample selected from a flitch of Eucalyptus pilularis (After Ghali et a/1979).

From each available position, thin hand-sections were made and examined from the transverse, radial and tangential wood faces as before. Some flitches sampled had fewer and some more sample blocks than shown in Fig. 10.

4.4 Results The results are summarised in Table 6.

17

4.5 Discussion From Table 6 it can be seen that the samples were

chiefly heartwood, i.e., tyloses were present in most samples. Fungal hyphae were widespread in the wood of all five trees but there was no obvious relationship with any previously determined wood property.

There was no evidence of clamp connections (which indicate potential decay fungi) but these structures are

frequently absent and more reliance is placed on evidence of cell wall breakdown. No degrade of cell walls by decay fungi , soft rots or bacteria was seen and again no link could be established between presence of fungal hyphae, or heartwood crystals associated with such hyphae, and breakdown of cell wall structure. The fungi present were therefore probably pioneer organisms associated with the initiation of the decay processes described by Shigo (1967, 1979).

In many samples vessel and fibre lumina were partly or completely filled by dark deposits of unknown

composition. These appeared structureless and were not birefringent. They caused wood discolouration but their presence could not be related to any mechanical properties of the samples .

4.6 Conclusions No link could be established between physical and

mechanical properties of the samples and presence or absence of fungi , bacteria, heartwood crystals or amorphous cell deposits .

Table 6. Presence of fungi , tyloses , heartwood crystals and dark amorphous deposits in wood of re growth blackbutt.

Tree Sample Heartwood Poly- Amorphous No. Height Fungi Tyloses Crystals phenols Deposits

Top +++++' -++++ +++++ ++-++ -++++ Butt +-+-- ++++++ -++++- --+ -++++-

2 Top - NS-+ ++NS++ ++NS-+ ++NS++ ++NS++ Butt ++++-+ ++-+++ -+- ++++++ --+++-

3 Top -++-+ ++++- ++++- ++++- ++++-Butt 1111 111111 --+++t+ ++- -++++++

4 Top +-+- +++++ +++++ +++++ +++++ Butt +-++++ ++-+++ ++-+++ +++++-

6 Top +++++ +++++ -++++ ++-++ +++++ Butt -+--+++- 11111 -+-++ -++++++- +1111111

I Each symbol represents a sample position across a tree from bark to bark. NS means no sample was available.

S. PENCIL STREAK, STAIN AND DECAY IN E. NITENS

Pencil streak, stains and decays of the same general type as for E. obliqua and E. pilularis also occur in shining gum (E. nitens Maiden).

As part of an examination of the effects of such stains, the mechanical properties of four visual grades of shining gum were determined (Ghali 1981), and material from each grade was examined microscopically for evidence of biological degrade (Edwards 1979b).

S.l Material and Methods Four groups of samples were selected from impact

toughness test blocks after these had been broken on a Denison tension testing machine. These were classified into the following visual grades:-

Group A - Clear: Normal, clear, defect-free material without imperfections, but allowing sloping grain I in 15. Group B - Light Stain: Defect-free material, allowing sloping grain as in Group A, borer holes up to 2 mm diameter, but not exceeding 10 in any lOO cm2• Black stain distribution density less than I cm2 j 100 cm2.

Group C - Medium Stain: Limited slope of grain as above, borer holes up to 3 mm wide, but not exceeding 20 in any 100 cm2• Black stain distribution density between 1-2 cm2j 100 cm2•

18

Group D - Heavy Stain: Limited slope of grain as above, borer holes and stained areas in excess of Group C.

From each sample hand sections were made from the transverse and radial longitudinal faces. These were mounted in lactophenol aniline blue stain and examined at 600x for evidence of biological degrade.

S.2 Results The results obtained are shown in Table 7.

S.3 Discussion Ghali et al (1981) showed significant differences

between means of the impact toughness tests at a T value of 1.96 as follows :

Visual No grade stain

Mean (loul) 20.91

Light stain 18.96

Medium stain 18.24

Heavy stain 16.42

Before interpreting the biological results some qualifications must be borne in mind. The type of data obtained comes from a search for gross differences, such as a clearly graded series of decay intensities between each of the visual grades. This did not occur, and the results could not be quantified because:

(a) Both dark deposits in the lumina of wood cells and the presence of dark hyphae will cause stain. In these tests the relative contribution of each factor could not be determined . Further

Ill)

'~

Table 7. Occurrence of fungi and associated materials in unseasoned shining gum (Eucalyptus nitens) impact toughness (Denison) samples.

stain indicators decay indicators

visual No. of freq.

dark grade samples deposits

(A) N 9 no 54 %A 17

stain % Total 8

(B) N 10 light 53 %B 19 stain % Total 9

(C) N 28 medium 51 %C 55

stain % Total 26

(D) N 32 heavy 53 %D 60 stain % Total 29

the area sampled was minute in relation to the total stained area, which makes an accurate assessment of the total effect of the stain fungi difficult.

(b) Cell wall degrade was the only reliable quantitative measure of decay available in this study. The presence of clamps indicates that potential decays were present but whether they were active, or could grow further in the wood after pioneer organisms had prepared the way was not determined. Again some of the hyaline and dark hyphae which were seen to be without clamps could be potential decay fungi since clamps are not always produced.

(c) A number of samples in all groups contained fungal fruiting bodies in the lumen of the vessels. These were Phialophora m elin ii, a Trichocladium sp. and a Ceratocystis sp. Phialophora spp. are common pioneer fungi and also saprophytes of plant material and they occur in soil. They are often found in association with dark stains of wood and wood pulp and may occur in black zones around borer holes. There is no evidence that they cause significant wood decay. Whether these were in the wood before felling or entered after sawing and storage is not known. Much of the hyphae present may have been of these species but this could not be determined microscopically. Ceratocystis spp. commonly cause blue stain in timber and are widespread in slash on the forest floor. Trichocladium spp. include pioneer species (Shigo, 1967).

With these points in mind it is possible however to draw some very broad inferences.

• Dark deposits in the cells appear to be correlated with the degree of stain as assessed by visual grading but not as well as with the mechanical properties. There was little difference between no­stain and light-stain grades or between medium

19

dark cell wall clamps hyaline

hyphae degrade hyphae

14 1 0 8 26 2 0 15 12 1 0 7

13 0 2 10 25 0 4 19 12 0 2 9

12 3 1 16 24 6 2 31 11 3 1 15

27 5 2 36 51 9 4 68 24 5 2 32

and heavy stain. Differences between the first two and last two groups were marked.

• The presence of dark hyphae appeared to be quantitatively indistinguishable in the groups, no­stain, light stain, and medium-stain, but there was a large difference between these groups and heavy stain. This may be due to severe insect attack with its related dark fungal hyphae. Again there was no obvious link with mechanical properties.

• Small differences (less than 10%) between samples showing decay are probably not significant in tests of this type since the decay was confined to a few cells in each sample and was not made up of large significant patches of damaged wood. A link between mechanical characteristics of these samples and the number showing signs of decay would be difficult to establish.

• A link is possible between the number of samples showing hyaline hyphae and those with dark deposits. However it is not known whether the dark hyphae contributed to the dark deposits.

5.4 Conclusions On the microscopic evidence available there did not

appear to be any correlation between either visual grades or impact toughness tests and the presence of decay, nor was there any evidence of a relationship between visual grading and the presence of staining fungi and dark wood-cell deposits.

6. DARK HEART IN TURPENTINE The incidence of the condition known as dark heart

of turpentine is often associated with defective wood by timber users. Following an examination of the mechanical properties of three trees of turpentine (Syncarpia glomulifera (Sm.) Niedenzu) showing this defect Ghali (1978) 25 Izod samples were tested for the presence of microbial deterioration. These samples are described in Table 8.

Table 8. Details of izod samples taken from three turpentine trees felled in the Karuah River Management Area. (Modified from Ghali, 1978.)

Tree Date Tree Tree No. Felled Height Dia. Billet

Code

(m) (cm)

1. 10.8.78 20 44 A

B C

2. -7.78 27 95 D

E

3. -7.78 24 52 H

Ghali (1978) concluded from his study that 'the influence of the discoloration upon the impact properties of turpentine is obvious and distinguishable' and that further study of the problem was importinit.

A loss of impact strength associated with dark stains of wood is sometimes associated with wood decay. To test this hand sections were cut fro.m each sample, stained in lactophenol aniline blue mountant and examined at 600x under normal and polarized light.

With one exception (sample D2), no pioneer, staining, soft rotting or decny-causing fungi could be detected. No evidence was seen of cell degrade, due either to these organisms, or to bacteria. There was thus no detectable difference between the red and dark brown samples due to microorganisms. Sample D2 showed slight traces of an unidentified fungus, but only in a few cells. No identifiable damage could be associated with it.

There appeared to be a greater deposition of materials in the vessels of the dark brown samples than in the red specimens, but the composition of this material was not determined. Incipient deterioration of the cell wall could not be detected by polarization methods because of the nature of the samples.

The lack of fungal hyphae or bacteria does not mean that they are not involved but the factors involved may be very complex, e.g. root rots, fire-induced biochemical changes, diffusion of bacterial or fungal enzymes, or ageing. However no convincing case could. be made to justify the considerable time and effort which would be needed to solve this problem.

I Ghali, M. Forestry Commission of New South Wales Internal Memorandum.

20

Billet Details

Sample Billet Billet Billet Samples Nos. Dia. Height Colour with

Above Sapwood Ground

(cm) (m)

Al-5 44 Red to AI, A5 Pink

Bl-4 36 4 B1, B4 Cl-4 32 8

" Cl, C4

Dl-2 N.A. 5 Dark D1 Brown

El-6 67 5 "

El, E6

Hl-5 44 5 Dark HI, H5 Brown

Ghalil has questioned whether brown heart in turpentine is a form of false heartwood. We have no evidence to confirm or disprove this.

False heartwood is a well known phenomenon (lane 1970; Panshin and de Zeeuw 1970; Perelvgin 1965). It occurs in a number of northern hemisphere species, e.g. beech, birch, maple, poplar and pines and may be dark or red brown, lilac, violet or dark green in colour. The vessels within the false heartwood area are heavily tylosed. The reasons for its formation are not clear, but may range from minor changes in the tree's metabolism which result in the deposition of coloured extractives in the wood and induce tyloses, up to more deep seated changes due to the invasion of fungal hyphae.

There are however similar staining problems not necessarily associated with false heartwood. Shigo (1979) describes the processes of discolouration and decay as a complex continuum of events that merge and overlap in time and place. He describes a sequence of events leading from biochemical changes due to injury, to invasion by bacteria and non-decay fungi, and finally attack by decay fungi. Discolourations which arise from such causes may occur after heartwood is formed. A typical example ofthe latter is brown stain of E. obliqua due to incipient decay.

7. ACKNOWLEDGEMENTS The assistance of Mrs. J. L. Pryjma and Mrs. V. E.

Magor who assisted in the laboratory work and Mr. R. Humphreys, Dr. R. K. Bamber and Mr. K. Bootle who made valuable comments on the text is gratefully acknowledged; as is Mr. R. Johnson formerly of the Royal Prince Alfred Hospital who took the insect photographs.

I .)

I

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APPENDIX 1

Forester's Classification of Eucalyptus obJiqua Test Logs Cut at Banshea State Forest

LOG No. LENGTH GIRTH FAULTS FAULTS FAULTS (cm) (cm) (BUTT) (TOP) (LENGTH)

1 488 158 Moderate Blk.* Heavy Blk. 2 549 137 Yz Dia. Blk. 12/16 Heavy Blk. 3 641 132 Heavy Black

8 Points Brittle Heavy Blk. 4 488 170 16/23 Moderate 14/19 Heavy Blk. 5 641 160 Heavy Blk. Heavy Black 6 366 86 Moderate Blk. Clear 10 point Occ.

Limb 7 427 127 Brittle Heart Brittle Heart

5 pts. 5 pts. 8 366 127 Clear Heavy Black 9 732 92 Gum 1 per 3 Gum 1 per 3

10 427 153 Moderate Blk. Heavy Blk. 11 366 168 Slight Black Heavy Blk.

No Registration ' 12 366 86 Clear 13 366 163 Slight Blk. Clear 1-? Gum Bllmp

No Registration 14 488 137 Outer Gum Outer Gum 1 Occ. Limb

15 pts. 15 549 153 Clear Brittle

Gum 1 per 2" 16 305 165 Clear Heavy Black 17 427 158 Gum 1 per 3 Moderate Black Epicormics

1 Scar. 12" 18 427 221 Heavy Black Heavy Blk. 1 Slight Swell

(Excess) (Excess) (Not accounted for)

19 366 188 Ants 2W' Heavy Black Spiral Grain (Excess) Gum Bumps

20 549 153 Brittle 15 pts. Brittle 5 pts. Scar 15 pts. Gum 3 pts. Limb 5 pts.

Epicormics 21 549 125 Black Excess Black (Excess) 1 Limb Ants

30 Epicormics 22 793 130 Brittle Brittle 2 Limbs 30 & 10

8 pts. Epics Top 10' 23 549 122 Black Brittle Clean

Black Gum 24 366 275 Black Rot 15 pts. Clear

Brittle 25 366 231 Black Black Green Epics.

*Blk = Black stain or pencilling Occ = Occluded Ants = Termites

24

'-,

I1 I,

I, I

APPENDIX 2

Culture Media

1. Sawdust medium for sporophore production Sawdust (Pinus radiata D. Don sapwood) Sawdust (Brachychiton acerifolium F. Muel!. sapwood) 360 g Maize meal 240 g Bone meal 15 g Potato starch 4.5 g Dried yeast 1.2 g Malt extract 3.0 g

I'~ Casein hydrolysate 1.05 g Tryptone 1.0 g Thiamine hydrochloride 0.02 g Tap water 720 ml

2. Malt/Yeast extract agar Malt extract 109 Glucose 20 g Yeast extract ("Vegemite") 10 g Agar ("Oxoid") 25 g Water 1000 ml

3. Oxidase tests for wood decays

• Nobles gum guaiac solution Gum guaiac 0.5 g 95% Alcohol 30 ml

• Bavendamn oxidase test agar for white and brown rots Malt extract 5g Agar (Oxoid) 20 g Gallic or tannic acid 5g Water 1000 ml

I

4. Stains • Lactophenol aniline blue for detection of fungi in wood

Phenol 20 g Lactic acid 16 ml Glycerol 31 ml Water 20 ml Aniline blue 0.05 %

25

APPENDIX 3

Cultural and other characteristics of basidiomycetes isolated from Eucalyptus spp.

Character 511 518 705 2841 3047 3417

1. Cultural appearance Advancing zone Even + + + + + + Bayed + Raised + + + + ~

Appressed + Growth rate Slow + Moderate + + Fast + + + Colony appearance Cottony + + + + + Downy + + + Felty + Floccose + Plumose + + Silky + Subfelty + Woolly + + + Chamois-like + Velvety +

2. Hyphal features

Hyphae Contorted + Encrusted + Fibre-like + Helicoid + + Interlocking + + Clamps + + + + Multiple Clamps + Monomitic Dimitic + + + + Trimitic + + Hyaline + + + + + + Coloured + + Contorted/ irregular + + Contorted/ encrusted + Spores Conidia + + + Oidia + +? Chlamydospores + + + +

Terminal + + + + Intercalary +

26

Character 511 518 705 2841 3047 3417

3. Biochemical Crystals present + + + + + Tannic Acid + + + + + Gallic Acid + + + + U.V. Melzers reagent

4. Odour 511 Sweet 518 Sour to citrus

'I 705 Nil 2841 Mushroom-like 3047 Banana-like 3417 Nil

5. Colour Edge Centre Reverse 511 White White Pale cream 518

" Pale straw Pale yellow

705 "

White Pale cream 2841

" " " 3047 "

Pale yellow " 3417

" Pale brown (saya1 zones) Dark brown

6. Pigments in media 3417 brown

7. Species from which isolated 511 E. obliqua 518

" 705 " 2841 E. pilularis

3047 " 3417 "

27