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Inoculation of Eucalyptus diversicolor thinning stumps with wood decay fungi for control of Armillaria luteobubalina
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MYCO~.Res. 94 (1): 32-37 (1990) Printed in Great Britain
Inoculation of Eucalyptus diuersicolor thinning stumps with wood decay fungi for control of Armillaria luteobubalina
M. H. PEARCE',' A N D N. MALAJCZUK2 'Soil Science and Plant Nutrition Group, School of Agriculture, University of Western Australia, Nedlands, W.A., 6009, Australia, and Division of Forestry and Forest Products, CSIRO, Private Bag, P.O. Wembley, 6014, Australia
Inoculation of Eucalpytus diversicolor thinning stumps with wood decay fungi for control of Amillaria luteobubalina. Mycological Research 94 (1):32-37 (1990). Eucalyptus diversicolor thinning stumps were simultaneously inoculated with the pathogen Amillaria luteobubalina and one of Coriolus versicolor, Stereum hirsutum and Xylaria hypoxylon to determine if these saprotrophic wood decay fungi could prevent or limit colonization of stumps by A. luteobubalina. The extent of fungal colonization 2 yr after inoculation was assessed at three positions in the stumps (10 cm above ground level, at ground level and 15 an below ground level). The three fungi equally significantly reduced colonization of stumps by A. luteobubalina, at each of the positions. C. versicolor decayed the upper portions of stumps most and X. hypoxylon least. Fmit-body production by the test fungi was significantly correlated to the relative wood density of the above ground portions of stumps. Some stumps were also colonized below ground by a naturally occurring cord-forming Hypholoma sp., resulting in complete or partial exclusion of A. luteobubalina and the test fungi from lower portions of those stumps. This fungus may prove more useful than the test fungi for control of A. luteobubalina. Key words: Amillaria luteobubalina, Stump inoculation, Biocontrol. Armillaria luteobubalina Watling & Kile is the most pathogenic and widespread of five Armillaria spp. described from Australia, causing dieback and death of a wide range of plant species in eucalypt forests, orchards, vineyards, parks and domestic gardens (Kile, 1981; Smith & Kile, 1981; Kile et al., 1983; Shearer & Tippett, 1988; Pearce, Malajczuk & Kile, 1986; Kile & Watling, 1988). In south-western Australia, forest tree hosts include karri (Eucalyptus diversicolor F. Muell.), a major sawlog species, with root rot being more severe in young regrowth stands than in older forest (Pearce ef al., 1986). Below-ground transmission of A. luteobubalina is mainly by root contact (Podger ef al., 1978; Kile, 1981; Pearce ef al., 1986), with infection of regrowth seedlings and saplings being closely associated with nearby infected stumps left in situ after logging operations (Kile, 1981; Pearce ef al., 1986). The few rhizomorphs produced by the fungus grow only for short distances in forest soils (Podger ef al., 1978; Kile, 1981; Pearce et al., 1986), and initiation of new infection centres by airborne basidiospores is relatively rare (Kile, 1983). Accordingly, control measures against A. luteobubalina need to be directed at limiting development of the fungus in stumps and associated roots. A number of control measures have been used against Arrnillaria spp., but physical removal of stumps and large roots (Doepel, 1962; Roth, Rolph & Cooley, 1980), soil fumigation around infected hosts or direct injection of Reprint requests to CSIRO address.
fumigants into infected hosts (Filip & Roth, 1977; Munnecke et al., 1981) are the most frequently advocated methods. However, these can be prohibitively costly and the use of fumigants toxic to other soil micro-organisms, fauna and stump decay fungi may also be environmentally unacceptable (Schiitt, 1985). An alternative method of control is by the inoculation of stumps with saprotrophic wood decay fungi which may either prevent or reduce Armillaria colonization of stumps (Rishbeth, 1976). Similar biocontrol measures have either been proven or are being tested world-wide for the control of other wood root-rot fungi, such as Heferobasidion annosum (Fr.) Bref., Phellinw noxius (Comer) G. H . Cunn., Phellinus weirii (Murr.) Gilb. and Chaefoporus radulus (Pers.: Fr.) Bond. & Singer (= Poria vincta (Berk.) Cke) (Rishbeth, 1963; Holdenrieder, 1984; Bolland, 1985; Nelson & Thies, 1985, 1986). This paper reports the results of a field trial to assess the feasibility of biocontrol of A. luteobubalina in karri thinning stumps using selected wood decay fungi.
MATERIALS A N D METHODS Fungal strains Four test strains (one of Coriolus versicolor (L.: Fr.) QuCI.; two of Stereurn hirsuturn (Willd.: Fr.) S. F. Gray; one of Xylaria hypoxylon (L.: Fr.) Grev.) were used. C. versicolor (MP. 111, M. Pearce), S. hirsuturn (MP. 65, M. Pearce) and X. hypoxylon (MP.
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M. H. Pearce and N. Malajczuk 29, M. Pearce) were obtained from fruiting bodies collected from karri stumps during 1983. The second S. hirsuturn strain was provided by Dr G. C. Johnson (Division of Forestry and Forest Products, CSIRO, Highett, Victoria) which was listed as Sfereum complicafum Fr. (Highett Accession No. 3967). This strain was recently identified as S. hirsuturn, based on the morphology of its basidiocarps produced on karri wood and on the cultural characteristics of monosporous isolates derived hom the basidiocarps (Dr A. M. Ainsworth, University of Bath, England, pers. comm.). These strains were selected because they actively or passively inhibited the growth of A. lufeobubalina in interspecific interaction tests on agar media and on wood blocks (Pearce & Malajczuk, 1983; Pearce, unpubl.). Study site
The study site (80 x 100 m) was located in an 8-yr-old karri regrowth forest near Pine Creek, ca 30 km west-south-west of Manjimup, Western Australia (lat. 34O 14' S, long. 116' 9' E). The forest had regenerated following clear-felling of mature forest and slash burning. The site is situated in a mid-slope position in a dissected laterite landscape. The soil is a red earth (according to Stace ef al., 1968) with a sandy loam surface gradually changing to a clay subsoil at depth. The region has cool, wet winters and hot, dry summers, with an annual rainfall of ca 1200 mm. Preparation of inoculum
Ramin (Gonystylus sp.) dowelling rods (6 cm x 1 cm diam) were soaked in 2% malt extract solution for 24 h, autoclaved for 30 min, placed onto 3 % malt extract agar in 9 cm diam Petri dishes, and inoculated with two 1cm2 agar discs of the test isolates. For the Armillaria inoculum, 6 cm x 3 un diam stem sections from freshly felled 3-yr-old karri were autoclaved for 1h in 2 1 flasks with 100 ml of distilled water. After cooling, several of the stem sections in each flask were inoculated with A. lufeobubalina agar discs. The sealed agar plates and flasks were incubated in darkness at 20 OC for 10 wk, when the dowels and stem sections were thoroughly permeated with mycelium. Stlamp inoculations
In May 1984, 35 suppressed karri saplings were felled leaving a horizontal stump surface, 6.5-11-0 an diam and 20-30 cm high. Stumps were immediately inoculated with a single test fungus by inserting the colonized dowels into holes drilled to a depth of 6 cm (one vertical hole at the centre of the cut surface, and three holes equidistant around the stump and near the base, drilled downwards at an angle of ca 45O towards the stump centre). Control stumps not inoculated with the test fungi were left intact (sterile dowels were specifically not inoculated into these stumps to compare a method of controlling A. lufeobubalina, via stump inoculation, as against a 'do-nothing' approach to control, as currently practiced in the kam regrowth forests). There were seven replicates of each treatment.
One major lateral root of each stump was inoculated with A. lufeobubalina 2-3 an from the root crown by removing a section 6 cm long and replacing it with an A. hteobubalina stem section to simulate spread of the fungus via root contact. Soil was replaced over the inoculated root and numbered metal tags were placed near each stump for identification. Coppice regrowth was removed from stumps at regular intervals until March 1985, after which no further growth occurred. Sampling methods and assessment of colonization
The presence of fungal fruiting bodies on the stumps was monitored monthly from Feb. 1985 to June 1986. In June 1986 the stumps were extracted with as much of the root system intact as was possible. To obtain a threedimensional view of h g a l colonization of stumps, the stumps were sectioned longitudinally into quarters and transversely into five sections (at 5 cm below stump tops, 10 cm above ground level (a.g.l.), ground level and 15 cm below ground level (b.g.1.)) on a band saw. The position and type of decayed or discoloured zones were recorded, and small wood chips from these zones were plated onto 3% malt extract agar (+amended with 8 p.p.m. benomyl, 8 p.p.m. dichloran and 50 p.p.m. polyphenol) to correlate decay with the presence of various fungi (Rayner, 1977a, b, 1979). To determine the effect of the test fungi on stump colonization by A. lufeobubalina, the percentage of stump diameter colonized by A. lufeobubalina was measured at three levels (10 cm a.g.l., at ground level and 15 cm b.g.1.) after removal of the bark layer. This percentage was based on the mean of two measurements at right angles to each other (from the four longitudinal stump sections) for each level. Radial sections of stumps (at 5 cm below stump tops, at ground level at 15 cm b.g.1.) were oven dried, measured and weighed to estimate relative wood density (and hence relative decay abilities of the test fungi). The oven dry weight of the h i t bodies produced on each stump was also measured to determine the correlation between relative wood density and hit-body production. Statistical analysis
Analyses of variance were carried out on the raw data of the percentage colonization and relative density values. Also as percentage colonization by A. luteobubalina varied from 0 to 70%, an angular transformation of the percentage data was undertaken and an analysis of variance was carried out on the transformed values. The conclusions from this analysis were not significantly different from the raw data analysis.
RESULTS All three test fungi significantly reduced, but did not prevent, stump colonization by A. lufeobubalina at each of the three levels (10 cm a.g.l., ground level and 15 c m b.g.l.), with no significant difference between the fungi (Fig. I). A. luteobubalina colonization of sapwood and bark was least at 10 cm a.g.1. and greatest at I 5 cm b.g.1. for the inoculated and control stumps.
Biocontrol of Armillaria lufeobubalina
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Fig. 1. A. luteobubalina colonization of karri thinning stumps 2 yr
basidiocarps had formed on four of the seven inoculated after inoculation with selected wood decay fungi. a,control stumps. 5. hirsufum basidiocarps first formed in July-Aug. (inoculated with A. luteobubalina only);e,Xylaria hypoxylon; 0, 1985 on four stumps. In early Mar. 1986, basidiocarp Coriolus versicolor; Stereum hirsutum (isolate A); 0 , S. hirsutum formation resumed on those four stumps, and by June 1986 (isolate B). L.s.D., Least significant difference (*P < 0-05, ***P< S. hirsufum had fruited on 13 of the 14 inoculated stumps. By 0.001). contrast, X. hypoxylon fruit bodies appeared on only one inoculated stump, in June 1986. The test fungi were re-isolated from sapwood of all respective inoculated stumps. There was a significant correlation between fruit-body production (oven L.S.D. dry weight) and relative density of above-ground portions of inoculated stumps (P < 0.02, r = 0.47, n = 26, for relative density near stump tops; and P < 0.05, r = 0.40, n = 26, for the mean of the relative densities at ground level and near stump tops). L.S.D. Armillaria hfeobubalina fruited on only three stumps (two control stumps and one S. hirsufum inoculated stump), in May-June 1986, despite being present in portions of the bark 00 I I and sapwood of all stumps. 10 cm above Ground 15 cm below An unidentified Hypholoma species (aff. H. capnoides ground level level ground level (Fr.:Fr.) Kummer, fide Dr 0.K. Miller Jr, pers. comm.) fruited Position on stump at the base of three stumps (I x control, I x 5. hirsufum and 1x X. hypoxylon inoculated stumps) in May-June 1986. At harvest, some Hypholoma basidiomes had fruited from one end Fig. 2. Relative decay of kam thinning slumps 2 yr after inoculation of an A. lufeobubalina inoculum block (at the base of the with selected wood decay fungi. a,control (inoculated with previously mentioned S. hirsutum-treated stump). The HyphoA. luteobubalina only); a,Xylaria hypoxylon; 0, Coriolus versicolor; loma sp. had colonized only the outer bark tissue and a Stereum hirsutum (isolate A); 0, S. hirsutum (isolate B). L.s.D.,Least small portion of one end of the inoculum block, as verified significant difference (* P < 0.05, *** P < 0.001). from the results of isolations made from the block. The basidiomes developed from white mycelial cords (ca 0.5 1mm diam) which had colonized the surface of the inoculum block. Visual inspection of sectioned stumps and the results of 0.4 isolations from decay zones indicated that the Hypholoma sp. had actually colonized portions of the sapwood of 13 stumps (2 controls, 3 x C. versicolor, 2 x X. hypoxylon and 2 of each of 0.3 the two S. hirsufum isolate treated stumps). In three of those stumps (1x 5. hirsufum, 1x C. versicolor and 1 x X. hypoxylon) the Hypholoma had completely colonized the lower portions 0.2 - L.S.D. of the stumps, excluding A. lufeobubalina and the test fungi (***) from two stumps up to 12 cm b.g.l., and one stump up to ground level (apart from a 10 cm2 area colonized by A. lufeobubalina near the inoculated root; Fig. 3b). Partial exclusion (50% of stump diam) of A. lufeobubalina colonization 15 cm above Ground 15 cm below by Hypholoma occurred with three other stumps (1x ground level level ground level C. versicolor, 1x S. hirsufum and 1x X. hypoxylon) at either Position on stump 15 cm b.g.1. and/or at ground level (Fig. 3d). Due to the effect of this Hypholoma sp. in reducing stump colonization by A. lufeobubalina, the colonization data were re-analysed after The test fungi and A. lufeobubalina colonized some stumps to substituting seven missing values for the stumps in which at least 50 cm b.g.1. (the maximum stump depth recovered). Hypholoma occurred at 15 cm b.g.1. or at ground level. This For the relative density analysis, the fungus x position on analysis indicated no significant effect on the conclusions stump interaction was highly significant (P< 0.001, Fig. 2). concerning the effects of the test fungi on A. lufeobubalina C. versicolor decayed the upper portions of stumps most and colonization. X. hypoxylon least. All of the test fungi colonized portions of some of the Fruit-body production by the test fungi also varied greatly. major lateral roots. Of the 114 lateral root segments recovered For C. versicolor, basidiocarp primordia first formed in (up to 30 cm long and between 1-8 cm diam at the root Feb.-Mar. 1985 around inoculum dowels on three stumps, but crown and 0.5-3 cm distally) from the inoculated stumps, 54 they did not develop into mature fruit bodies during 1985. were colonized by A. lufeobubalina alone (these occurred Between Mar. and Apr. 1986, C. versicolor fruited prolifically approximately equally between treatments), 4 by the test on one additional stump and by harvest (in June 1986), fungi alone, 19 with A. luteobubalina and the test fungi (with
+,
+,
I
35
M. H. Pearce and N. Malajczuk
Fig. 3. Diagrams of de-barked stump cross-sections illustrating fungal colonizat~onof stumps 2 yr after inoculation: A, control (inoculated with A. luteobubalina only; clear zone colonized by an unidentified fungus); B, Xylaria hypoqlon-inoculated stump, with below-ground colonization by a Hypholoma sp.; C. Conolw versicolor-inoculated stump; D, Stereurn hinuturn-inoculated stump, showing partial colonization by the Hypholoma sp. O, wood colonized by A. luteobubahna; M, wood colonized by a Hypholoma s p . ; a , uncolonized heart wood; 0, uncolonized dead wood; 12,dowel inoculurn plug; Psp, black zone line between A. luteobubalina and other fungi; Clear zone in B, C and D, wood colonized by the inoculated test fungi.
A . hteobubalina in the bark and outer sapwood and the test
fungi in the more central wood of roots), 18 were colonized by Hypholoma alone, 7 with A . luteobubalina and Hypholoma, 2 with Hypholoma and the test fungi, and 10 roots were not colonized (the 7 roots on one stump were still alive, as was most of the below-ground portion of that stump, and the other 3 roots were on separate stumps). For the control stumps, 26 roots were colonized by A . luteobubalina alone, 2 with Hypholoma alone and one with A. luteobubalina and
~~~holoma.
Typical patterns of fungal colonization are shown in Fig. 3. Black zone lines ('pseudosclerotial plates', commonly produced by A . luteobubalina (Podger et al., 1978; Kile, 1981; Pearce et al., 1986) and by other Armillaria species (Mallet & Hiratsuka, 1986)), always separated A . luteobubalina colonization areas and tissues colonized by other fungi; but where A . luteobubalina infection areas bordered either alive or dead, non-colonized inner sapwood, no zone lines occurred (Fig. 3 a ) . The above-ground inner sapwood of most of the control stumps was colonized by several unidentified fungi (due to spore colonization by air-borne spores) (Fig. 3 a ) . Xylaria hypoxylon was isolated (identity confirmed by production of typical fruit bodies on agar media) from portions of above- and below-ground sapwood and bark of two control stumps, from one lateral root of an S. hirsutum inoculated stump and from a small portion of the belowground sapwood of a C. versicolor inoculated stump. Natural colonization of stumps by X. hypoxylon can occur aerially or
from below ground, the latter probably via germination of dormant ascospores in close proximity to stumps (Coates & Rayner, 1985 a, b).
DISCUSSION This study has indicated that inoculation of stumps with selected wood decay fungi can significantly reduce stump colonization by A . luteobubalina. The three test fungi, C. versicolor, S. hirsutum and X. hypoxylon, colonized aboveground and below-ground sapwood of stumps, including some major lateral roots. However, below-ground colonization by those fungi did not prevent A. luteobubalina from colonizing most of the below-ground outer sapwood and inner bark of most treated stumps, due to the ability of A . luteobubalina to colonize stumps rapidly by subcortical mycelial growth, an ability not shared by the test fungi. There was no significant difference between C. versicolor, S. hirsutum and X. hypoxylon in reducing A. luteobubalina colonization of stumps. Also, there was little evidence of active replacement of A. lufeobubalina by the three test fungi. It is therefore evident that the main mode of action by the test fungi in reducing Armillaria inoculum levels in stumps was by their prior possession of stump tissues (excepting the cambial region) and their ability not to be replaced by A. luteobubalina, both important attributes of biocontrol fungi (Rishbeth, 1976). In this study, an unidentified Hypholoma sp. fortuitously
Biocontrol of Armillaria lufeobubalina colonized portions of several stumps, in some cases excluding A. luteobubalina from below-ground portions of stumps (including major lateral roots), more s o than the test fungi. Cord-forming wood decay fungi such as Hypholoma, Phanerochaefe and Phlebia species have a similar niche to Armillaria, which may be important in relation to biological control of Armillaria (Dowson, Rayner & Boddy, 1988a-c; Rayner, 1977 b, 1979). This is because if establishment of the pathogen is to b e prevented then any potential competitor should come into early contact with it in stump tissues, which is most likely with fungi sharing Armillaria's capacity for subcortical mycelial growth and ability t o colonize roots, otherwise competition may be avoided until a late stage (Rayner, 1979). Field observations of this unidentified Hypholoma sp. in several karri forest sites indicate that the fungus produces mycelial cords, it is not pathogenic and it colonizes the outer dead bark of roots of living trees (M. Pearce, unpubl.), thus being in a positional advantage t o quickly colonize roots of freshly cut stumps. The attributes of this fungus therefore indicate that it may b e a more suitable organism t o use in biocontrol against A. hteobubalina than fungi such as C. versicolor, S. hirsufum and X. hypoxylon. Inoculation of thinning stumps with rapid-decay organisms could have other beneficial effects as well as contributing t o Armillaria control. These may include rapid stump removal to improve site access for future management operations and increased rates of nutrient turnover in the forest. Thinning stumps might also be inoculated with edible fungi such as shiitake (Lentinula edodes (Berk.) Pegler) which may provide a cash crop from an otherwise untapped resource. Nevertheless, our study indicates that the routine inoculation of thinning stumps with suitable fungi over successive crop rotations in plantation forests would significantly reduce the incidence of A. luteobubalina in those forests.
DOWSON, C. G., RAYNER, A. D. M. & BODDY, L. (1988~). The form and outcome of mycelial interactions involving cord-forming decomposer basidiomycetes in homogeneous and heterogeneous environments. New Phytologist 109, 423-432. FILIP, G. M. & ROTH, L. F. (1977). Stump injections with soil fumigants to eradicate Armillariella mellea from young-growth ponderosa pine killed by root rot. Canadian Iournal of Forest Research 7, 226-231. HOLDENRIEDER, VON 0 . (1984). Untersuchungen zur biologischen Bekampfung von Heterobasidion annosum an Fichte (Picea abies) mit antagonistischen Pilzen. 11. Interaktionstests auf Holz. European Iournal of Forest Pathology 14, 137-153. KILE, G. A. (1981). Armillaria luteobubalina: a primary cause of decline and death of trees in mixed species eucalypt forests in central Victoria. Australian Forest Research 11,63-77. KILE, G. A. (1983). Identification of genotypes and the clonal development of Armillaria luteobubalina Watling & Kile in eucalypt forests. Australian Journal of Botany 31,657-671. KILE, G. A. & WATLING, R. (1988). Identification and occurrence of Australian Amillaria species including A. pallidula sp. nov. and comparative studies between them and non-Australian tropical and Indian Armillaria. Transactions of the British Mycological Society 91,305-315. KILE, G. A,, WATLING, R., MALAJCZUK, N. & SHEARER, B. L. (1983). Occurrence of Amillaria luteobubalina Watling & Kile in Western Australia. Australasian Plant Pathology 12, 18-20. MALLETT, K. I. & HIRATSUKA, Y. (1986). Nature of the 'black line' produced between different biological species of the Armillaria mellea complex. Canadian Iournal of Botany 64,2588-2590. McARTHUR, W. M. & CLIFTON, A. L. (1975). Forestry and agriculture in relation to soils in the Pemberton area of Western Australia. CSIRO (Australia) Division of Soils, Soil and Land Use Sen'es No. 54. MUNNECKE, D. E., KOLBEZEN, M. J., WILBUR, W. D. & OHR, H. D. (1981). Interactions involved in controlling Amillaria mellea. Plant Disease 65,384-389. NELSON, E. E. & THIES, W. G. (1985). Colonization of Phellinus weirii-infested stumps by Trichoderma viride. I. Effect of isolate and inoculum base. European Journal of Forest Pathology 15, 425The authors thank Linda Pearce and Dr Earl Nelson for 431. assistance in setting u p and harvesting the trial respectively, E. E. & THIES, W. G. (1986). Colonization of Phellinw NELSON, and Drs L. Bolland and G. A. Kile for constructive criticism of weirii-infested stumps by Trichoderma viride. 2. Effects of season of the manuscript. inoculation and stage of wood decay. European Journal of Forest Pathology 16, 56-60. REFERENCES PEARCE, M. H. & MALAJCZUK. N. (1983). Interactions between Amillaria luteobubalina and wood decaying basidiomycetes of the BOLLAND, L. (1985). Biocontrol of root-rot in Hoop Pine - pilot kam (Eucalyptus diversicolor) forests of south-westem Australia. In stump inoculation study. Department of Forestry, Queensland, Abstracts of Papers, 4th International Congress of Plant Pathology, Research Results No. 1 of 1985. COATES, D & RAYNER, A. D. M. (1985 a). Fungal population and Aug. 1983, p. 242. North Melbourne, Australia: Rowprint Services community development in cut beech logs I. Establishment via the (Vic.) Pty Ltd. aerial cut surface. New Phytologist 101, 153-171. PEARCE, M. H., MALAJCZUK, N. & KILE, G. A. (1986). The COATES, D. & RAYNER, A. D. M. (1985 b). Fungal population and occurrence and effects of Armillaria luteobubalina in the kam community development in cut beech logs. 11. Establishment via (Eucalyptus diversicolor F. Muell.) forests of Western Australia. the buried cut surface. New Phytologist 101, 173-181. Australian Forest Research 16,243-259. DOEPEL, R. F. (1962). Armillaria root rot of fruit trees. Western PODGER, F. D., KILE, G. A., WATLING, R. & FRYER, J. (1978). Australian Journal of Agriculture (Fourth Series) 3, 39-42. Spread and effects of Armillaria luteobubalina sp. nov. in an DOWSON, C. G., RAYNER, A. D. M. & BODDY, L. (1988~). Australian Eucalyptus regnans plantation. Transactions of the British Inoculation of mycelial cord-forming basidiomycetes into woodMycological Society 71, 77-87. land soil and litter. I. Initial establishment. New Phytologist 109, RAYNER, A. D. M. (1977~).Fungal colonization of hardwood 335-341. stumps from natural sources. I. Non-basidiomycetes. Transactions of the British Mycological Society 69,291-302. DOWSON, C. G., RAYNER, A. D. M. & BODDY, L. (19886). Inoculation of mycelial cord-forming basidiomycetes into woodRAYNER, A. D. M. (1977b). Fungal colonization of hardwood land soil and litter. 11. Resource capture and persistence. New stumps from natural sources 11. Ba~idiom~cetes. Transactions of the Phytologist 109, 343-349. British Mycological Society 69,303-312.
M. H. Pearce and N. Malajczuk RAYNER, A. D. M. (1979).Internal spread of fungi inoculated into hardwood stumps. New Phyfologisf 8 2 , 505-517. RISHBETH, J. (1963).Stump pr,otection against Fornes annosw. 111. Inoculation with Peniophora gigantea. Annals of Applied Biology 5 2 , 63-77. RISHBETH, J. (1976). Chemical treatment and inoculation of hardwood stumps for control of Armillaria mellea. Annals of Applied Biology 8 2 , 57-70. ROTH, L. F., ROLPH, L. & COOLEY, S. (1980).Identifying infected Ponderosa Pine stumps to reduce costs of controlling Armillaria root rot. ]ournu1 of Forestry 78, 145-151.
37 SCHUTT, P. (1985).Control of root and butt roots: limits and prospects. European ]ournu1 of Forest Pathology 15, 357-363. SHEARER, B. L. & TIPPETT, J. T. (1988).Distribution and impact of Armillaria lufeobubalinain the Eucalyptus marginata forest of southwestern Australia. Australian ]oumul of Botany 36, 433-445. SMITH, L. & KILE, G. A. (1981).Distribution and hosts of Armillaria root rot in Melbourne suburban gardens. Australasian Plant Pathology 10, 41-42. STACE, H. C. T., HUBBLE, G . D., BREWER, R., NORTHCOTE, K. H., SLEEMAN, J. R., MULCAHY, M. J. & HALLSWORTH, E. G. (1968).A Handbook of Australian Soils. Glenside, South Australia: Rellim Technical Publications.
(Received for publication 22 December 1988)
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MYCO~.Res. 94 (1): 32-37 (1990) Printed in Great Britain
Inoculation of Eucalyptus diuersicolor thinning stumps with wood decay fungi for control of Armillaria luteobubalina
M. H. PEARCE',' A N D N. MALAJCZUK2 'Soil Science and Plant Nutrition Group, School of Agriculture, University of Western Australia, Nedlands, W.A., 6009, Australia, and Division of Forestry and Forest Products, CSIRO, Private Bag, P.O. Wembley, 6014, Australia
Inoculation of Eucalpytus diversicolor thinning stumps with wood decay fungi for control of Amillaria luteobubalina. Mycological Research 94 (1):32-37 (1990). Eucalyptus diversicolor thinning stumps were simultaneously inoculated with the pathogen Amillaria luteobubalina and one of Coriolus versicolor, Stereum hirsutum and Xylaria hypoxylon to determine if these saprotrophic wood decay fungi could prevent or limit colonization of stumps by A. luteobubalina. The extent of fungal colonization 2 yr after inoculation was assessed at three positions in the stumps (10 cm above ground level, at ground level and 15 an below ground level). The three fungi equally significantly reduced colonization of stumps by A. luteobubalina, at each of the positions. C. versicolor decayed the upper portions of stumps most and X. hypoxylon least. Fmit-body production by the test fungi was significantly correlated to the relative wood density of the above ground portions of stumps. Some stumps were also colonized below ground by a naturally occurring cord-forming Hypholoma sp., resulting in complete or partial exclusion of A. luteobubalina and the test fungi from lower portions of those stumps. This fungus may prove more useful than the test fungi for control of A. luteobubalina. Key words: Amillaria luteobubalina, Stump inoculation, Biocontrol. Armillaria luteobubalina Watling & Kile is the most pathogenic and widespread of five Armillaria spp. described from Australia, causing dieback and death of a wide range of plant species in eucalypt forests, orchards, vineyards, parks and domestic gardens (Kile, 1981; Smith & Kile, 1981; Kile et al., 1983; Shearer & Tippett, 1988; Pearce, Malajczuk & Kile, 1986; Kile & Watling, 1988). In south-western Australia, forest tree hosts include karri (Eucalyptus diversicolor F. Muell.), a major sawlog species, with root rot being more severe in young regrowth stands than in older forest (Pearce ef al., 1986). Below-ground transmission of A. luteobubalina is mainly by root contact (Podger ef al., 1978; Kile, 1981; Pearce ef al., 1986), with infection of regrowth seedlings and saplings being closely associated with nearby infected stumps left in situ after logging operations (Kile, 1981; Pearce ef al., 1986). The few rhizomorphs produced by the fungus grow only for short distances in forest soils (Podger ef al., 1978; Kile, 1981; Pearce et al., 1986), and initiation of new infection centres by airborne basidiospores is relatively rare (Kile, 1983). Accordingly, control measures against A. luteobubalina need to be directed at limiting development of the fungus in stumps and associated roots. A number of control measures have been used against Arrnillaria spp., but physical removal of stumps and large roots (Doepel, 1962; Roth, Rolph & Cooley, 1980), soil fumigation around infected hosts or direct injection of Reprint requests to CSIRO address.
fumigants into infected hosts (Filip & Roth, 1977; Munnecke et al., 1981) are the most frequently advocated methods. However, these can be prohibitively costly and the use of fumigants toxic to other soil micro-organisms, fauna and stump decay fungi may also be environmentally unacceptable (Schiitt, 1985). An alternative method of control is by the inoculation of stumps with saprotrophic wood decay fungi which may either prevent or reduce Armillaria colonization of stumps (Rishbeth, 1976). Similar biocontrol measures have either been proven or are being tested world-wide for the control of other wood root-rot fungi, such as Heferobasidion annosum (Fr.) Bref., Phellinw noxius (Comer) G. H . Cunn., Phellinus weirii (Murr.) Gilb. and Chaefoporus radulus (Pers.: Fr.) Bond. & Singer (= Poria vincta (Berk.) Cke) (Rishbeth, 1963; Holdenrieder, 1984; Bolland, 1985; Nelson & Thies, 1985, 1986). This paper reports the results of a field trial to assess the feasibility of biocontrol of A. luteobubalina in karri thinning stumps using selected wood decay fungi.
MATERIALS A N D METHODS Fungal strains Four test strains (one of Coriolus versicolor (L.: Fr.) QuCI.; two of Stereurn hirsuturn (Willd.: Fr.) S. F. Gray; one of Xylaria hypoxylon (L.: Fr.) Grev.) were used. C. versicolor (MP. 111, M. Pearce), S. hirsuturn (MP. 65, M. Pearce) and X. hypoxylon (MP.
33
M. H. Pearce and N. Malajczuk 29, M. Pearce) were obtained from fruiting bodies collected from karri stumps during 1983. The second S. hirsuturn strain was provided by Dr G. C. Johnson (Division of Forestry and Forest Products, CSIRO, Highett, Victoria) which was listed as Sfereum complicafum Fr. (Highett Accession No. 3967). This strain was recently identified as S. hirsuturn, based on the morphology of its basidiocarps produced on karri wood and on the cultural characteristics of monosporous isolates derived hom the basidiocarps (Dr A. M. Ainsworth, University of Bath, England, pers. comm.). These strains were selected because they actively or passively inhibited the growth of A. lufeobubalina in interspecific interaction tests on agar media and on wood blocks (Pearce & Malajczuk, 1983; Pearce, unpubl.). Study site
The study site (80 x 100 m) was located in an 8-yr-old karri regrowth forest near Pine Creek, ca 30 km west-south-west of Manjimup, Western Australia (lat. 34O 14' S, long. 116' 9' E). The forest had regenerated following clear-felling of mature forest and slash burning. The site is situated in a mid-slope position in a dissected laterite landscape. The soil is a red earth (according to Stace ef al., 1968) with a sandy loam surface gradually changing to a clay subsoil at depth. The region has cool, wet winters and hot, dry summers, with an annual rainfall of ca 1200 mm. Preparation of inoculum
Ramin (Gonystylus sp.) dowelling rods (6 cm x 1 cm diam) were soaked in 2% malt extract solution for 24 h, autoclaved for 30 min, placed onto 3 % malt extract agar in 9 cm diam Petri dishes, and inoculated with two 1cm2 agar discs of the test isolates. For the Armillaria inoculum, 6 cm x 3 un diam stem sections from freshly felled 3-yr-old karri were autoclaved for 1h in 2 1 flasks with 100 ml of distilled water. After cooling, several of the stem sections in each flask were inoculated with A. lufeobubalina agar discs. The sealed agar plates and flasks were incubated in darkness at 20 OC for 10 wk, when the dowels and stem sections were thoroughly permeated with mycelium. Stlamp inoculations
In May 1984, 35 suppressed karri saplings were felled leaving a horizontal stump surface, 6.5-11-0 an diam and 20-30 cm high. Stumps were immediately inoculated with a single test fungus by inserting the colonized dowels into holes drilled to a depth of 6 cm (one vertical hole at the centre of the cut surface, and three holes equidistant around the stump and near the base, drilled downwards at an angle of ca 45O towards the stump centre). Control stumps not inoculated with the test fungi were left intact (sterile dowels were specifically not inoculated into these stumps to compare a method of controlling A. lufeobubalina, via stump inoculation, as against a 'do-nothing' approach to control, as currently practiced in the kam regrowth forests). There were seven replicates of each treatment.
One major lateral root of each stump was inoculated with A. lufeobubalina 2-3 an from the root crown by removing a section 6 cm long and replacing it with an A. hteobubalina stem section to simulate spread of the fungus via root contact. Soil was replaced over the inoculated root and numbered metal tags were placed near each stump for identification. Coppice regrowth was removed from stumps at regular intervals until March 1985, after which no further growth occurred. Sampling methods and assessment of colonization
The presence of fungal fruiting bodies on the stumps was monitored monthly from Feb. 1985 to June 1986. In June 1986 the stumps were extracted with as much of the root system intact as was possible. To obtain a threedimensional view of h g a l colonization of stumps, the stumps were sectioned longitudinally into quarters and transversely into five sections (at 5 cm below stump tops, 10 cm above ground level (a.g.l.), ground level and 15 cm below ground level (b.g.1.)) on a band saw. The position and type of decayed or discoloured zones were recorded, and small wood chips from these zones were plated onto 3% malt extract agar (+amended with 8 p.p.m. benomyl, 8 p.p.m. dichloran and 50 p.p.m. polyphenol) to correlate decay with the presence of various fungi (Rayner, 1977a, b, 1979). To determine the effect of the test fungi on stump colonization by A. lufeobubalina, the percentage of stump diameter colonized by A. lufeobubalina was measured at three levels (10 cm a.g.l., at ground level and 15 cm b.g.1.) after removal of the bark layer. This percentage was based on the mean of two measurements at right angles to each other (from the four longitudinal stump sections) for each level. Radial sections of stumps (at 5 cm below stump tops, at ground level at 15 cm b.g.1.) were oven dried, measured and weighed to estimate relative wood density (and hence relative decay abilities of the test fungi). The oven dry weight of the h i t bodies produced on each stump was also measured to determine the correlation between relative wood density and hit-body production. Statistical analysis
Analyses of variance were carried out on the raw data of the percentage colonization and relative density values. Also as percentage colonization by A. luteobubalina varied from 0 to 70%, an angular transformation of the percentage data was undertaken and an analysis of variance was carried out on the transformed values. The conclusions from this analysis were not significantly different from the raw data analysis.
RESULTS All three test fungi significantly reduced, but did not prevent, stump colonization by A. lufeobubalina at each of the three levels (10 cm a.g.l., ground level and 15 c m b.g.l.), with no significant difference between the fungi (Fig. I). A. luteobubalina colonization of sapwood and bark was least at 10 cm a.g.1. and greatest at I 5 cm b.g.1. for the inoculated and control stumps.
Biocontrol of Armillaria lufeobubalina
34
Fig. 1. A. luteobubalina colonization of karri thinning stumps 2 yr
basidiocarps had formed on four of the seven inoculated after inoculation with selected wood decay fungi. a,control stumps. 5. hirsufum basidiocarps first formed in July-Aug. (inoculated with A. luteobubalina only);e,Xylaria hypoxylon; 0, 1985 on four stumps. In early Mar. 1986, basidiocarp Coriolus versicolor; Stereum hirsutum (isolate A); 0 , S. hirsutum formation resumed on those four stumps, and by June 1986 (isolate B). L.s.D., Least significant difference (*P < 0-05, ***P< S. hirsufum had fruited on 13 of the 14 inoculated stumps. By 0.001). contrast, X. hypoxylon fruit bodies appeared on only one inoculated stump, in June 1986. The test fungi were re-isolated from sapwood of all respective inoculated stumps. There was a significant correlation between fruit-body production (oven L.S.D. dry weight) and relative density of above-ground portions of inoculated stumps (P < 0.02, r = 0.47, n = 26, for relative density near stump tops; and P < 0.05, r = 0.40, n = 26, for the mean of the relative densities at ground level and near stump tops). L.S.D. Armillaria hfeobubalina fruited on only three stumps (two control stumps and one S. hirsufum inoculated stump), in May-June 1986, despite being present in portions of the bark 00 I I and sapwood of all stumps. 10 cm above Ground 15 cm below An unidentified Hypholoma species (aff. H. capnoides ground level level ground level (Fr.:Fr.) Kummer, fide Dr 0.K. Miller Jr, pers. comm.) fruited Position on stump at the base of three stumps (I x control, I x 5. hirsufum and 1x X. hypoxylon inoculated stumps) in May-June 1986. At harvest, some Hypholoma basidiomes had fruited from one end Fig. 2. Relative decay of kam thinning slumps 2 yr after inoculation of an A. lufeobubalina inoculum block (at the base of the with selected wood decay fungi. a,control (inoculated with previously mentioned S. hirsutum-treated stump). The HyphoA. luteobubalina only); a,Xylaria hypoxylon; 0, Coriolus versicolor; loma sp. had colonized only the outer bark tissue and a Stereum hirsutum (isolate A); 0, S. hirsutum (isolate B). L.s.D.,Least small portion of one end of the inoculum block, as verified significant difference (* P < 0.05, *** P < 0.001). from the results of isolations made from the block. The basidiomes developed from white mycelial cords (ca 0.5 1mm diam) which had colonized the surface of the inoculum block. Visual inspection of sectioned stumps and the results of 0.4 isolations from decay zones indicated that the Hypholoma sp. had actually colonized portions of the sapwood of 13 stumps (2 controls, 3 x C. versicolor, 2 x X. hypoxylon and 2 of each of 0.3 the two S. hirsufum isolate treated stumps). In three of those stumps (1x 5. hirsufum, 1x C. versicolor and 1 x X. hypoxylon) the Hypholoma had completely colonized the lower portions 0.2 - L.S.D. of the stumps, excluding A. lufeobubalina and the test fungi (***) from two stumps up to 12 cm b.g.l., and one stump up to ground level (apart from a 10 cm2 area colonized by A. lufeobubalina near the inoculated root; Fig. 3b). Partial exclusion (50% of stump diam) of A. lufeobubalina colonization 15 cm above Ground 15 cm below by Hypholoma occurred with three other stumps (1x ground level level ground level C. versicolor, 1x S. hirsufum and 1x X. hypoxylon) at either Position on stump 15 cm b.g.1. and/or at ground level (Fig. 3d). Due to the effect of this Hypholoma sp. in reducing stump colonization by A. lufeobubalina, the colonization data were re-analysed after The test fungi and A. lufeobubalina colonized some stumps to substituting seven missing values for the stumps in which at least 50 cm b.g.1. (the maximum stump depth recovered). Hypholoma occurred at 15 cm b.g.1. or at ground level. This For the relative density analysis, the fungus x position on analysis indicated no significant effect on the conclusions stump interaction was highly significant (P< 0.001, Fig. 2). concerning the effects of the test fungi on A. lufeobubalina C. versicolor decayed the upper portions of stumps most and colonization. X. hypoxylon least. All of the test fungi colonized portions of some of the Fruit-body production by the test fungi also varied greatly. major lateral roots. Of the 114 lateral root segments recovered For C. versicolor, basidiocarp primordia first formed in (up to 30 cm long and between 1-8 cm diam at the root Feb.-Mar. 1985 around inoculum dowels on three stumps, but crown and 0.5-3 cm distally) from the inoculated stumps, 54 they did not develop into mature fruit bodies during 1985. were colonized by A. lufeobubalina alone (these occurred Between Mar. and Apr. 1986, C. versicolor fruited prolifically approximately equally between treatments), 4 by the test on one additional stump and by harvest (in June 1986), fungi alone, 19 with A. luteobubalina and the test fungi (with
+,
+,
I
35
M. H. Pearce and N. Malajczuk
Fig. 3. Diagrams of de-barked stump cross-sections illustrating fungal colonizat~onof stumps 2 yr after inoculation: A, control (inoculated with A. luteobubalina only; clear zone colonized by an unidentified fungus); B, Xylaria hypoqlon-inoculated stump, with below-ground colonization by a Hypholoma sp.; C. Conolw versicolor-inoculated stump; D, Stereurn hinuturn-inoculated stump, showing partial colonization by the Hypholoma sp. O, wood colonized by A. luteobubahna; M, wood colonized by a Hypholoma s p . ; a , uncolonized heart wood; 0, uncolonized dead wood; 12,dowel inoculurn plug; Psp, black zone line between A. luteobubalina and other fungi; Clear zone in B, C and D, wood colonized by the inoculated test fungi.
A . hteobubalina in the bark and outer sapwood and the test
fungi in the more central wood of roots), 18 were colonized by Hypholoma alone, 7 with A . luteobubalina and Hypholoma, 2 with Hypholoma and the test fungi, and 10 roots were not colonized (the 7 roots on one stump were still alive, as was most of the below-ground portion of that stump, and the other 3 roots were on separate stumps). For the control stumps, 26 roots were colonized by A . luteobubalina alone, 2 with Hypholoma alone and one with A. luteobubalina and
~~~holoma.
Typical patterns of fungal colonization are shown in Fig. 3. Black zone lines ('pseudosclerotial plates', commonly produced by A . luteobubalina (Podger et al., 1978; Kile, 1981; Pearce et al., 1986) and by other Armillaria species (Mallet & Hiratsuka, 1986)), always separated A . luteobubalina colonization areas and tissues colonized by other fungi; but where A . luteobubalina infection areas bordered either alive or dead, non-colonized inner sapwood, no zone lines occurred (Fig. 3 a ) . The above-ground inner sapwood of most of the control stumps was colonized by several unidentified fungi (due to spore colonization by air-borne spores) (Fig. 3 a ) . Xylaria hypoxylon was isolated (identity confirmed by production of typical fruit bodies on agar media) from portions of above- and below-ground sapwood and bark of two control stumps, from one lateral root of an S. hirsutum inoculated stump and from a small portion of the belowground sapwood of a C. versicolor inoculated stump. Natural colonization of stumps by X. hypoxylon can occur aerially or
from below ground, the latter probably via germination of dormant ascospores in close proximity to stumps (Coates & Rayner, 1985 a, b).
DISCUSSION This study has indicated that inoculation of stumps with selected wood decay fungi can significantly reduce stump colonization by A . luteobubalina. The three test fungi, C. versicolor, S. hirsutum and X. hypoxylon, colonized aboveground and below-ground sapwood of stumps, including some major lateral roots. However, below-ground colonization by those fungi did not prevent A. luteobubalina from colonizing most of the below-ground outer sapwood and inner bark of most treated stumps, due to the ability of A . luteobubalina to colonize stumps rapidly by subcortical mycelial growth, an ability not shared by the test fungi. There was no significant difference between C. versicolor, S. hirsutum and X. hypoxylon in reducing A. luteobubalina colonization of stumps. Also, there was little evidence of active replacement of A. lufeobubalina by the three test fungi. It is therefore evident that the main mode of action by the test fungi in reducing Armillaria inoculum levels in stumps was by their prior possession of stump tissues (excepting the cambial region) and their ability not to be replaced by A. luteobubalina, both important attributes of biocontrol fungi (Rishbeth, 1976). In this study, an unidentified Hypholoma sp. fortuitously
Biocontrol of Armillaria lufeobubalina colonized portions of several stumps, in some cases excluding A. luteobubalina from below-ground portions of stumps (including major lateral roots), more s o than the test fungi. Cord-forming wood decay fungi such as Hypholoma, Phanerochaefe and Phlebia species have a similar niche to Armillaria, which may be important in relation to biological control of Armillaria (Dowson, Rayner & Boddy, 1988a-c; Rayner, 1977 b, 1979). This is because if establishment of the pathogen is to b e prevented then any potential competitor should come into early contact with it in stump tissues, which is most likely with fungi sharing Armillaria's capacity for subcortical mycelial growth and ability t o colonize roots, otherwise competition may be avoided until a late stage (Rayner, 1979). Field observations of this unidentified Hypholoma sp. in several karri forest sites indicate that the fungus produces mycelial cords, it is not pathogenic and it colonizes the outer dead bark of roots of living trees (M. Pearce, unpubl.), thus being in a positional advantage t o quickly colonize roots of freshly cut stumps. The attributes of this fungus therefore indicate that it may b e a more suitable organism t o use in biocontrol against A. hteobubalina than fungi such as C. versicolor, S. hirsufum and X. hypoxylon. Inoculation of thinning stumps with rapid-decay organisms could have other beneficial effects as well as contributing t o Armillaria control. These may include rapid stump removal to improve site access for future management operations and increased rates of nutrient turnover in the forest. Thinning stumps might also be inoculated with edible fungi such as shiitake (Lentinula edodes (Berk.) Pegler) which may provide a cash crop from an otherwise untapped resource. Nevertheless, our study indicates that the routine inoculation of thinning stumps with suitable fungi over successive crop rotations in plantation forests would significantly reduce the incidence of A. luteobubalina in those forests.
DOWSON, C. G., RAYNER, A. D. M. & BODDY, L. (1988~). The form and outcome of mycelial interactions involving cord-forming decomposer basidiomycetes in homogeneous and heterogeneous environments. New Phytologist 109, 423-432. FILIP, G. M. & ROTH, L. F. (1977). Stump injections with soil fumigants to eradicate Armillariella mellea from young-growth ponderosa pine killed by root rot. Canadian Iournal of Forest Research 7, 226-231. HOLDENRIEDER, VON 0 . (1984). Untersuchungen zur biologischen Bekampfung von Heterobasidion annosum an Fichte (Picea abies) mit antagonistischen Pilzen. 11. Interaktionstests auf Holz. European Iournal of Forest Pathology 14, 137-153. KILE, G. A. (1981). Armillaria luteobubalina: a primary cause of decline and death of trees in mixed species eucalypt forests in central Victoria. Australian Forest Research 11,63-77. KILE, G. A. (1983). Identification of genotypes and the clonal development of Armillaria luteobubalina Watling & Kile in eucalypt forests. Australian Journal of Botany 31,657-671. KILE, G. A. & WATLING, R. (1988). Identification and occurrence of Australian Amillaria species including A. pallidula sp. nov. and comparative studies between them and non-Australian tropical and Indian Armillaria. Transactions of the British Mycological Society 91,305-315. KILE, G. A,, WATLING, R., MALAJCZUK, N. & SHEARER, B. L. (1983). Occurrence of Amillaria luteobubalina Watling & Kile in Western Australia. Australasian Plant Pathology 12, 18-20. MALLETT, K. I. & HIRATSUKA, Y. (1986). Nature of the 'black line' produced between different biological species of the Armillaria mellea complex. Canadian Iournal of Botany 64,2588-2590. McARTHUR, W. M. & CLIFTON, A. L. (1975). Forestry and agriculture in relation to soils in the Pemberton area of Western Australia. CSIRO (Australia) Division of Soils, Soil and Land Use Sen'es No. 54. MUNNECKE, D. E., KOLBEZEN, M. J., WILBUR, W. D. & OHR, H. D. (1981). Interactions involved in controlling Amillaria mellea. Plant Disease 65,384-389. NELSON, E. E. & THIES, W. G. (1985). Colonization of Phellinus weirii-infested stumps by Trichoderma viride. I. Effect of isolate and inoculum base. European Journal of Forest Pathology 15, 425The authors thank Linda Pearce and Dr Earl Nelson for 431. assistance in setting u p and harvesting the trial respectively, E. E. & THIES, W. G. (1986). Colonization of Phellinw NELSON, and Drs L. Bolland and G. A. Kile for constructive criticism of weirii-infested stumps by Trichoderma viride. 2. Effects of season of the manuscript. inoculation and stage of wood decay. European Journal of Forest Pathology 16, 56-60. REFERENCES PEARCE, M. H. & MALAJCZUK. N. (1983). Interactions between Amillaria luteobubalina and wood decaying basidiomycetes of the BOLLAND, L. (1985). Biocontrol of root-rot in Hoop Pine - pilot kam (Eucalyptus diversicolor) forests of south-westem Australia. In stump inoculation study. Department of Forestry, Queensland, Abstracts of Papers, 4th International Congress of Plant Pathology, Research Results No. 1 of 1985. COATES, D & RAYNER, A. D. M. (1985 a). Fungal population and Aug. 1983, p. 242. North Melbourne, Australia: Rowprint Services community development in cut beech logs I. Establishment via the (Vic.) Pty Ltd. aerial cut surface. New Phytologist 101, 153-171. PEARCE, M. H., MALAJCZUK, N. & KILE, G. A. (1986). The COATES, D. & RAYNER, A. D. M. (1985 b). Fungal population and occurrence and effects of Armillaria luteobubalina in the kam community development in cut beech logs. 11. Establishment via (Eucalyptus diversicolor F. Muell.) forests of Western Australia. the buried cut surface. New Phytologist 101, 173-181. Australian Forest Research 16,243-259. DOEPEL, R. F. (1962). Armillaria root rot of fruit trees. Western PODGER, F. D., KILE, G. A., WATLING, R. & FRYER, J. (1978). Australian Journal of Agriculture (Fourth Series) 3, 39-42. Spread and effects of Armillaria luteobubalina sp. nov. in an DOWSON, C. G., RAYNER, A. D. M. & BODDY, L. (1988~). Australian Eucalyptus regnans plantation. Transactions of the British Inoculation of mycelial cord-forming basidiomycetes into woodMycological Society 71, 77-87. land soil and litter. I. Initial establishment. New Phytologist 109, RAYNER, A. D. M. (1977~).Fungal colonization of hardwood 335-341. stumps from natural sources. I. Non-basidiomycetes. Transactions of the British Mycological Society 69,291-302. DOWSON, C. G., RAYNER, A. D. M. & BODDY, L. (19886). Inoculation of mycelial cord-forming basidiomycetes into woodRAYNER, A. D. M. (1977b). Fungal colonization of hardwood land soil and litter. 11. Resource capture and persistence. New stumps from natural sources 11. Ba~idiom~cetes. Transactions of the Phytologist 109, 343-349. British Mycological Society 69,303-312.
M. H. Pearce and N. Malajczuk RAYNER, A. D. M. (1979).Internal spread of fungi inoculated into hardwood stumps. New Phyfologisf 8 2 , 505-517. RISHBETH, J. (1963).Stump pr,otection against Fornes annosw. 111. Inoculation with Peniophora gigantea. Annals of Applied Biology 5 2 , 63-77. RISHBETH, J. (1976). Chemical treatment and inoculation of hardwood stumps for control of Armillaria mellea. Annals of Applied Biology 8 2 , 57-70. ROTH, L. F., ROLPH, L. & COOLEY, S. (1980).Identifying infected Ponderosa Pine stumps to reduce costs of controlling Armillaria root rot. ]ournu1 of Forestry 78, 145-151.
37 SCHUTT, P. (1985).Control of root and butt roots: limits and prospects. European ]ournu1 of Forest Pathology 15, 357-363. SHEARER, B. L. & TIPPETT, J. T. (1988).Distribution and impact of Armillaria lufeobubalinain the Eucalyptus marginata forest of southwestern Australia. Australian ]oumul of Botany 36, 433-445. SMITH, L. & KILE, G. A. (1981).Distribution and hosts of Armillaria root rot in Melbourne suburban gardens. Australasian Plant Pathology 10, 41-42. STACE, H. C. T., HUBBLE, G . D., BREWER, R., NORTHCOTE, K. H., SLEEMAN, J. R., MULCAHY, M. J. & HALLSWORTH, E. G. (1968).A Handbook of Australian Soils. Glenside, South Australia: Rellim Technical Publications.
(Received for publication 22 December 1988)
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