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Effects of the cord-forming saprotrophs Hypholoma australe and Phanerochaete filamentosa and of ammonium sulphamate on establishment of Armillaria luteobubalina on stumps of Eucalyptus diversicolor
Mycol. Res. 99 (8), 951-956 (1995)
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Printed in Great Britain
Effects of the cord-forming saprotrophs Hypholoma australe and Phanerochaete filamentosa and of ammonium sulphamate on establishment of Armillaria luteobubalina on stumps of Eucalyptus diversicolor
M. H. PEARCE 1 ,,., E. E. NELS OW AND N. MALAJCZUK 1 1 2
Division of Forestry, CSIRO, Private Bag, P.O. Wembley, WA., 6014, Australia USDA, Forest Service, Pacific Northwest Research Station, Forestry Sciences Laboratory, 3200 Jefferson Way, Corvallis, Oregon, 97331, U.SA.
Freshly cut karri (Eucalyptus diversicolor) thinning stumps were simultaneously inoculated with the pathogen Armillaria luteobubalina and either Hypholoma australe or Phanerochaete filamentosa. The stumps were inoculated with H. australe and P. filamentosa either above- or below-ground, and treated with either 40% (w/v) aqueous ammonium sulphamate solution (AMS) or water. Fungal colonization 2 yr after inoculation was assessed. AMS treatment had several highly significant effeds. Without AMS, neither biocontrol agent significantly reduced Armillaria colonization. However, with AMS, both P. filamentosa and H. australe significantly reduced Armillaria colonization, P. filamentosa being more effedive. Below-ground inoculation was more effedive in reducing Armillaria colonization than above-ground inoculation. In the absence of the biocontrol agents, AMS increased below-ground colonization by Armillaria compared with nonAMS treated stumps, but reduced colonization at and above ground level. Stump decay was increased, and coppice occurrence reduced, with AMS treatment. The naturally-occurring fungi Stereum hirsutum and Trametes versicolor fruited only on AMS stumps, whereas Chondrostereum purpureum fruited only on non-AMS stumps. Fruiting by Hypholoma australe was enhanced by AMS, whereas Armillaria luteobubalina fruiting was significantly greater on non-AMS stumps.
Armillaria species cause root and butt rot of many economically important forest and horticultural tree species worldwide (Shaw & Kile, 1991). Stumps are commonly colonized by the pathogen, leading to infection of nearby trees. A number of control measures have been used against Armillaria spp. However, the methods advocated most frequently, e.g. physical removal of stumps and large roots (Roth, Rolph & Cooley, 1980), and soil fumigation around infected hosts or direct injection of fumigants into infected hosts (Filip & Roth, 1977; Munnecke et a/., 1981) can be prohibitively costly in forests. Moreover, the use of fumigants toxic to other soil micro-organisms, fauna and stump decay fungi may be environmentally unacceptable (Schutt, 1985). An alternative method may be to inoculate stumps with saprotrophic wood decay fungi that either prevent or reduce Armillaria colonization (Rishbeth, 1976; Pearce & Malajczuk, 1990 a). Pearce & Malajczuk (1990a) found that although inoculation of eucalypt stumps with Trameles versicolor (L. :Fr.) Pilcit, Stereum hirsutum (Willd.: Fr.) Pers. and Xylaria hypoxylon (L.: Fr.) Grev. significantly reduced the volume of stumps colonized by Armillaria luteobubalina Watling & Kile, the test fungi were unable to colonize the subcortical region of stumps rapidly and so did not prevent establishment of Armillaria in this region. Cord-forming wood decay fungi such as Hypholoma and Phanerochaete species have a niche similar to Armillaria and share its capacity for rapid subcortical mycelial growth • Current address: 10 Goodall St, Lesmurdie, 6076, Western Australia.
(Rayner, 1977 b, 1979), and may be important in biological control (Dowson, Rayner & Boddy, 1988a, b, c; Rayner, 1977b, 1979; Pearce & Malajczuk, 1990a; Worrall, 1991). Also, chemical treatment of stumps, e.g. with ammonium sulphamate (AMS) may lead to rapid colonization of stumps by fungi which preclude entry of Armillaria (Rishbeth, 1976). This paper reports the results of a field trial in southwestern Australia to assess the efficacy of inoculating kani (Eucalyptus diversicolor F. Muel!.) thinning stumps with cordforming wood decay fungi, in combination with chemical (AMS) treatment, to control Armillaria luteobubalina biologically. A similar biocontrol study, undertaken concurrently but with antagonistic Trichoderma isolates, is reported separately (Nelson, Pearce & Malajczuk, 1995).
MATERIALS AND METHODS The experimental design included two test fungi, two stump inoculation techniques (above- and below-ground), two chemical treatments (AMS and water), and two slightly different control treatments. There were five replicates of each treatment. Fungal strains One strain each of Hypholoma australe nom. ined. (MP. 225, M. H. Pearce, and referred to in Pearce & Malajczuk, 1990a as Hypholoma sp.) and Phanerochaete filamentosa (Berk. & M. A.
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Biocontrol of Armillaria with cord-forming fungi Curtis) Burds. (MP. 226, M. H. Pearce) were used. The P. filamentosa strain was obtained from a mycelial cord attached to a fruit body on a Banksia grandis Willd. stump, in a jarrah (Eucalyptus marginata Donn. ex Sm.) forest site at Bickley, Western Australia, in 1984. The A. luteobubalina strain is described in Pearce & Malajzcuk (1990a).
inoculated with the test fungi were (a) stumps left intact (apart from the Armillaria inoculation), and (b) non-colonized dowels were inserted into drill holes similar to the AGL fungal inoculation treatments. One replicate of each treatment was included in each of the stump size groups.
Sampling methods and assessment of colonization Study site The study site (ca 4 ha) was located in an 11-yr-old karri regrowth forest near Pine Creek ca 30 km west-south-west of Manjimup, Western Australia (lat. 34° 14/ S, long. 116° 9/ E). Additional site data are provided by Pearce & Malajczuk (1990a). A. luteobubalina was present adjacent to the site, and in the same soil type (red earth).
Preparation of inoculum Two different types of test fungus inoculum were used. In one, Ramin (Gonystylus sp.) dowelling rods (6 cm xl cm diam.) were soaked in 2 % malt extract solution for 1 h, autoclaved at 121°C for 30 min, placed onto 3% malt extract agar in 9 cm diam. Petri plates, and inoculated with two 1 em 2 agar discs of the test isolates. In the other, 6 cm x 3 em diam. stem sections from freshly felled 3-yr-old karri were autoclaved for 1 h in 2 I flasks with 100mI of distilled water. After cooling, several of the stem sections in each flask were inoculated with agar discs of the test fungi. The Armillaria inoculum was also prepared by this second method. The sealed agar plates and flasks were incubated in darkness at 20° for 5 and 8-12 wk respectively, by which time the dowels and stem sections were thoroughly permeated with mycelium.
Stump inoculations In May (late autumn) 1987, 60 suppressed but otherwise healthy karri saplings were felled and stumps (8'0-11'2 cm diam. and 30 em high) cut horizontally, using a chain saw. Stumps were identified with an aluminium or stainless steel tag and galvanized naiL and were divided equally into five groups with cut surface diameters of 8'0-8'6,8'7-9'2,9'3-9'9, 10'0-10'6 and 10'7-11'2 cm. Half of each of the size groups of stumps were poisoned with 40% wjv ammonium sulphamate (AMS) applied in two separate applications of ca 20 ml, sufficient to cover the stump surface. A major lateral root on all stumps was inoculated with A. luteobubalina by removing a section 6 cm long at the root crown and replacing it with two inoculum sections against the fresh wound and packing them in place, to simulate spread of the fungus via root contact. The inoculated root was re-covered with soil. Stumps were inoculated with a single test fungus either (1) above ground level (AGL) by inserting the colonized dowels into three 1 cm diam. holes drilled to a depth of 6 cm eqUidistant around the stump and near the base (drilled downwards at an angle of ca 45° towards the stump centre), or (2) below ground level (BGL) by placing karri stem inoculum on a cut root on the opposite side of the stump to the A. luteobubalina inoculum, follOWing the same procedure as the A. luteobubalina inoculation. Control treatments not
The presence of basidiomes and coppice regrowth on stumps was regularly monitored dUring May and June of 1988 and from March to June in 1989. In June-July 1989 the stumps were excavated and removed with as much of the root system intact as was possible (in most cases there was a single taproot with smaller lateral roots). The stumps were transported to the laboratory, debarked and sectioned transversely at 10 cm intervals in either direction from the ground line on a band saw, and later split longitudinally into halves or quarters with a chisel. The top section, 20-30 em AGL, of each stump was discarded as it was not required for analysis. The three-dimensional positions and types of decayed or discoloured zones of the remaining sections were recorded, and representative small wood chips sampled from these zones were plated onto 3 % malt extract agar (with or without 30 ppm streptomycin sulphate) and onto the Goldfarb, Nelson & Hansen (1989) medium but supplemented with 30 ppm streptomycin sulphate and 30 ppm Rose Bengal, to correlate decay patterns with the presence of various fungi (Rayner 1977 a, b, 1979; Pearce & Malajczuk, 1990a). After sampling, some of these stump sections, particularly P. filamentosa treatments, were also incubated in sealed plastic bags' for 2-3 wk (to allow for fungal growth to occur) to assist in identification of decay zones. The percentage of stump diameter colonized by A. luteobubalina was measured at each of the above-mentioned 10 em intervals after removal of the bark layer, This percentage was based on the mean of two representative perpendicular diam. measurements for each level (as per Pearce & Malajczuk, 1990a).
Approximate wood density (as a measure of decay) was determined for stump sections (10-20 em AGL, 0-10 cm AGL, 0-10 em BGL and 10-20 em BGL) by displacement of water, drying for 3 wk at 70° and weighing.
Statistical analysis Analyses of variance were carried out on the raw data for the percentage colonization and wood density values. Also, as percentage colonization by A. luteobubalina varied from 0 to 62 %, 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. Also, analyses of variance were undertaken on the AMS data alone (i.e. excluding the non-AMS data) because of the overwhelming effects that AMS treatment had on the results, i.e. to determine if there were significant fungus treatment effects in the AMS data which may have been masked in the overall data analysis.
M. H. Pearce, E. E. Nelson and N. Malajczuk
953
Chi square analyses were undertaken to examine the effects of AMS on coppice regrowth and fruiting by naturally occurring and inoculated fungi.
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Coppice presence on stumps was greatly reduced by AMS application compared with the non-AMS (water treated) controls, particularly at the end of year 1 (Table 1, P < 0'001 for 1988 and P < 0-01 for 1989). The water treated stumps with live coppice occurred equally between both the five size groups of stumps, and the different stump treatment combinations, in 1988 and 1989. Fruiting on stumps by some naturally occurring fungi was significantly affected by AMS application. Stereum hirsutum fruited only on AMS treated stumps (on 13 stumps in 1988 and 20 in 1989), whereas Chondrostereum purpureum (Pers.:Fr.) Pouzar fruited only on non-AMS treated stumps (two in 1988 and six in 1989). Trametes versicolor fruited on two AMS stumps in 1989, but not on any non-AMS treated stumps. Hypholoma australe fruiting was enhanced by AMS treatment (X 2 = 3'28, 0'05 < P < 0'10, based on 1988/89 combined data). This fungus fruited on two Hypholomainoculated AMS stumps and one non-AMS stump in 1988 and on five AMS and one untreated stump in 1989. A. luteobubalina fruited only in 1989, more so on non-AMS stumps (eight) than on AMS stumps (two) (X 2 = 4'32, P < 0'05), despite being present in portions of the bark and/or sapwood of all stumps (but refer to isolations results below). In three cases, A. luteobubalina and H. austmle fruited on the same stump, with fruiting of each fungus occurring only on opposite sides of these stumps, corresponding with their respective colonization zones. The stump excavations indicated that P. filamentosa fruited on three stumps (two AMS and one non-AMS stumps), underneath the bark. Mycelial cords of this fungus were present in the soil around these stumps.
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Stump colonization by A. luteobubaIina and other fungi The all-data analysis (AMS and non-AMS treated stumps) indicated that whilst all main effects were significant (fungus P < 0'001, chemical P < 0'01, stump section P < 0·00l). Table 1. Effect of 40% aqueous ammonium sulphamate treatment of Eucalyptus diversicoloT sapling stumps on coppice regrowth, 1 and 2 yr after treatment No. of stumps with Year observed
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June 1988
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Position on stump (em) Fig. 1. Interaction between ammonium sulphamate (AMS) chemical treatment and stump section (above and below ground level) on colonization by Armillaria luteobubalina in Eucalyptus diversicolor sapling srumps. 0, AMS; non-AMS (water treated); AGL, above ground level; BGl, below ground level; bars, least significant difference (', P < 0'05, "', P < 0'001) for means of all treatments.
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there were two significant first order interactions (fungus x chemical P < 0'05, and chemical x stump section P < 0'001) which affected stump colonization by A. luteobubalina. There were no other significant interactions (at P < 0'05), and stump diameter had no significant effect on Armillaria colonization (at P < 0'05). With the chemical x stump section interaction, AMS Significantly (P < 0'001) enhanced BGL colonization by Armillaria compared with non-AMS stumps, whereas at GL (ground level) and AGL Armillaria colonization was reduced by AMS application (Fig. 1). With the fungus x chemical interaction, there was no significant fungus effect on Armillaria colonization with the non-AMS stumps, but there were some highly significant (P < 0'001) fungus effects with AMS treated stumps (Fig. 2). Phanerochaete filamentosa was more effective in reducing Armillaria colonization than was Hypholoma austmle, and BGL inoculation was more effective than AGL for both cordformers. The P. filamentosa-BGl inoculation combination was the only treatment which consistently greatly reduced (i.e. all but eliminated) Armillaria colonization at all stump levels measured, in comparison with all other treatments (Fig. 2). Armillaria colonization of the drill-control stumps (inoculated with non-colonized dowels) was slightly less than the control stumps left intact, particularly at 20 em BGl (P < 0'05 at 20 em BGl, Fig. 2). This may have been due to (a) accidental introduction of aerial colonists deeper into stump tissues during the drilling/dowel insertion process, and/or (b) more favourable micro-environmental conditions for growth of saprotrophs shortly after the drilling/dowel insertion process, i.e. more available oxygen deeper in the drilled stumps, compared with the intact stumps.
Biocontrol of Armillaria with cord-forming fungi
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Stump treatment Fig. 2. The effects of fungus treatment and stump section (at and below ground level) on colonization by Armillaria luteobubalina in Eucalyptus diversicolor sapling stumps treated with ammonium sulphamate. 0, at ground level; f:::., 10 em BGL; _, 20 cm BGL; bars, least significant difference (', P < 0'05; "', P < 0'001); p, PmmerocMete jilamentosa; H, Hypholoma australe; B, below ground inoculation; A, above ground inoculation; C control stumps left intact; CD, control stumps with non-colonized dowels. There was no colonization of stumps by A. luteobubalina at 10 or 20 em AGL.
The isolations from representative decay zones, together with visual assessments, indicated that fungal colonization was strongly influenced by ± AMS treatment. Firstly, the non-AMS treated stumps generally remained alive (particularly BGL), and for those non-AMS stumps inoculated with dowelling, colonization was strongly localized in most stumps around inoculation points. Secondly, and more specifically: (a) Chondroslereum purpureum was only isolated from nonAMS stumps (from 11/30 stumps), mostly from sections 2-4 (20 em AGL - 10 em BGL) but sometimes from sections 5-7, and occurred equally between the various stump treatments (as did all other naturally occurring fungi mentioned below). (b) Sterer-lm hirsulum occurred far more commonly, and more extensively, in AMS stumps than in non-AMS stumps (in 26/30 and 2/30 stumps respectively, and from all sections from AMS stumps, but only in sections 2-4 of non-AMS srumps). (c) Phanerochaele jilamentosa was successfully isolated from the sapwood of only one non-AMS treated stump (BGL inoculation), but from seven of the AMS stumps (five BGLand two AGL-inoculated stumps). For the five AMS BGLinoculated stumps, P. jilamentosa was isolated mostly from the external sapwood (which was extensively decayed), with the more inner sapwood colonized predominantly by S. hirsutum in four stumps and by T. versicolor in the fifth stump. (d) Hypholoma australe was re-isolated from 9/10 non-
AMS Hypholoma-inoculated stumps (from sections 2-5) and from all (10/10) AMS Hypholoma-inoculated stumps (from sections 2-7 in most cases). (e) Trameles versicolor was isolated from one non-AMS stump and from four AMS treated stumps, and occurred more extensively in the AMS stumps than in the non-AMS stumps. (f) Armillaria luteobubalina was present in the sapwood of 21/30 non-AMS stumps, and for the other nine non-AMS stumps, it had either not colonized below the bark or had only colonized small areas of the cambial region in the form of mycelial sheets and had not penetrated into the live sapwood (the latter also evidenced by the lack of any host reaction mechanisms, such as staining, in the sapwood). By contrast, A. luteobubalina was present in the sapwood of all AMS stumps. However, for the P. jilamentosa BGL-inoculated stumps, it was only present in traces as represented by small localized pockets in the outer sapwood, with the remainder of the sapwood colonized by saprotrophic decay fungi. Generally, the aerially-colonizing fungi (particularly S. hirsutum and to a lesser degree T. versicolor, and several infrequent unidentified fungi), were restricted mainly to the inner sapwood in belowground srump sections, with A. luleobubalina and/or the inoculated cord-forming fungi colonizing the outer sapwood. This was true also for most lateral root segments examined.
Stump decay Density of stump sections was significantly lower in AMStreated stumps thannon-AMS stumps (37'21 and 44'39 g cm- 3 respectively, LSD = 1'19, P < O·OOI). For the combined data analysis there was no significant main effect of fungus treatment on decay. However, the AMS-only data analysis indicated that the P. jilamenlosa and H. australe BGL-inoculated stumps were equally decayed significantly more than the Control and Drill Control stumps (P < 0'05), and that BGL inoculation resulted in greater decay than AGL inoculation for the two cord-forming fungi (P < 0'01 for P. jilamentosa and P < 0'05 for H. auslrale). Stump diam. significantly affected decay (P < 0'001 for combined data, and P < 0'05 for AMSonly data analyses respectively).
DISCUSSION This study has indicated that inoculation of stumps with cordforming wood decay fungi, combined with AMS treatment, significantly reduces stump colonization by A. luleobubalina, more so than the three non-cord-forming fungi tested by Pearce & Malajczuk (1990a). This is most likely because of the ability of H. australe and P. jilamenlosa to colonize stumps rapidly by subcortical mycelial growth, an ability not shared by the fungi tested by Pearce & Malajczuk (1990a), and hence negating Armillaria's normal positional advantage (wherein Armillaria can also rapidly colonize stumps by subcortical mycelial growth). The significantly greater effect of P. jilamentosa in reducing A. luteobubalina colonization of stumps more than H. australe is probably a reflection of P. jilamentosa's greater capacity for primary resource capture (Cooke & Rayner, 1984; Rayner & Webber, 1984). It has an intrinsic
M. H. Pearce, E. E. Nelson and N. Malajczuk ability to produce a more extensive network of mycelial cords than H. australe (Pearce & Malajczuk, 1990b; Pearce, unpublished data; and sensu Dowson et al., 1988a, b, c) with a resultant ability to colonize available domain by mycelial cords faster than H. australe. It is interesting to note, however, that in agar interactions (Pearce, 1990), H. australe (referred to therein as Hypholoma sp. A) is more combative against A. luteobubalina than is P. filamentosa. This apparent anomaly is easily explained, in that for a potential biocontrol organism to be successful. it does not need to be able to replace or kill the pathogen (although this would be desirable), but it only needs to occupy the niche and to resist replacement by the pathogen (sensu Rayner & Webber, 1984), which appears to be the situation in this study. The dramatic effect that AMS treatment had on reducing coppice regrowth and on enhancing colonization of hardwood stumps by naturally occurring fungi (as observed in this study) has been previously documented in the United Kingdom (Rishbeth, 1976; Rayner, 1977 a, b, 1979). Nevertheless, although AMS treatment quickly killed the stumps and markedly favoured above ground colonization of stumps by rapid decay fungi (5. hirsutum and to a lesser extent T. versicolor), all but eliminating Armillaria from above-ground sections of stumps, AMS enhanced below-ground colonization of stumps by A. Iuteobubalina (Fig. 1). This is probably due to Armillaria's positional advantage and ability to take advantage of the sudden (AMS-induced) decrease in host resistance to fungal infection. This positional advantage of A. luteobubalina limits the colonization of below-ground stump tissues by the aerial colonists to the more central sapwood (as observed in this study and by Pearce & Malajczuk, 1990a), thus enabling A. Iuteobubalina to continue to spread to new hosts via rootto-root contact. It is only when the fast-colonizing cordforming fungi, such as P. filamentosa and to a lesser extent H. australe, intervene that A. luteabubalina is significantly controlled, as they act by preventing A. luteobubalina from colonizing the outer sapwood. the pathogen's lifeline to a major foodbase. It is likely therefore, that a qualification to Rishbeth's 1976 study is required, in that whilst AMS application to stumps not already infected by Armillaria will probably prevent subsequent infection (because of rapid colonization of all sapwood by fungi such as 5. hirsutum and T. versicolor, and probably also by cord-forming fungi if they are present in the soil [sensu Dowson et al., 1988a, b, cD, it may not lead to adequate control if Armillaria is already present on the roots/root crown, sensu Swift's 1970 conclusions of the effects of ringbarking of trees on colonization by Armillaria. An important implication from this observation is that if AMS is to be used as a stump poison in Armillaria infected areas, then stumps will also need to be inoculated with suitable biocontrol (cord-forming) fungi, Le. an integrated control programme seems to be required for successful biocontrol of Armillaria. Otherwise, Armillaria may continue to be a significant problem in those areas. Notwithstanding the above, whilst AMS treatment significantly reduces AGL resource availability for A. luteobubalina (Fig. 1), one potential problem which may become evident is the necessity for the favoured cord-forming fungi (either
955
introduced or from natural colonization) to either resist replacement by these aerial colonizers, or if they are replaced, then these aerial colonists must in tum be able to either resist replacement by Armillaria, or replace Armillaria. Interaction studies to date indicate that S. hirsl,dum easily replaces P. filamentosa on agar, but it does not replace H. australe (M. H. Pearce, unpublished data). However, A. luteobubalina is unable to replace S. hirsutum or T. versicolor (Pearce, 1990). This study supports the views of Dowson et al. (1988a, b, c), Rayner (1977 b, 1979) and Pearce & Malajczuk (1990a), on the actual or potential biocontrol activity of cord-forming fungi against Armillaria spp. Also, an attractive attribute of using cord-forming fungi as biocontrol agents is that they can be readily established in woodland soils by direct seeding with colonized wood blocks (Dowson et al., 1988a, b, c; refer also Rayner & Boddy, 1988: 402). It is consequently envisaged that if the most efficient cord-formers are used (Le. those that quickly produce extensive cord systems in soil and subsequently rapidly colonize stump roots etc.), inoculation of individual stumps may not be required, which is most favourable from an economic viewpoint. We thank Stuart Eales, David Pearce, Robert Pearce, Tony Pearce, Dr Bernie Dell and Joanne Robinson for assistance in field operations, Carol Nelson for invaluable assistance in the laboratory, Dr Tim Grove for assistance with the statistical analyses, Dr P. K. Buchanan for verification of C. purpureum, and Dr O. K. Miller Jr for taxonomic study of Hypholoma australe. We gratefully acknowledge funding from a Reserve Bank of Australia Rural Credits Development Grant for part of this study. REFERENCES Cooke, R. C. & Rayner. A. D. M. (1984). Ecology of Saprotrophic Fungi. Longman: London. Dawson, C. G., Rayner, A. D. M. & Boddy, L. (1988a). Inoculation of mycelial cord-forming basidiomycetes into woodland soil and litter I. Initial establishment. New Phytalagist 109, 335-341. Dowson, C. G., Rayner, A. D. M. & Boddy, L. (1988 b). Inoculation of mycelial cord·forming basidiomycetes into woodland soil and litter. II. Resource capture and persistence. New Phytalagist 109, 343-349. Dowson, C. G., Rayner, A. D. M. & Boddy, L. (1988 c). The form and outcome of mycelial interactions involving cord·forming decomposer basidiomycetes in homogeneous and heterogeneous environments. New PhytaJogist 109, 423-432. Filip, G. M. & Roth, L. F. (1977). Stump injections with soil fumigants to eradicate Annillariel/a mel/ea from young-growth ponderosa pine killed by root rot. Canadian Journal of Forest Research 7, 226-231. Goldfarb, B.. Nelson, E. E. & Hansen, E. M. (1989). Trichodenna spp.:growth rates and antagonism to Phellinus weirii in vitro. Mycalogia 81, 375-381. Munnecke, D. E., Kolbezen, M.)., Wilbur, W. D. & Ohr, H. D. (I 9tH). Interactions involved in controlling Armillaria mel/ea. Plant Disease 65, 384-389.
Nelson, E. E., Pearce, M. H. & Malajczuk. N. (1995). Effects of Trichadenna spp. and ammonium sulphamate on establishment of Annil/aria luteobubalina on stumps of Eucalyptus diversicolar. Mycological Research 99, 957-962. Pearce, M. H. (1990). In vitro interactions between Annillaria luteobubalina and other wood decay fungi. Mycological Research 94, 753-761. Pearce, M. H. & Malajczuk, N. (1990a). Inoculation of karri (Eucalyptus diversicoJor F. Muell.) thinning stumps with wood decay fungi for control of Annillaria luteobubalina. Mycological Research 94, 32-37. Pearce, M. H. & Malajczuk, N. (1990b). Stump colonization by Annillaria luteobubalina and other wood decay fungi in an age series of cut-over
BiocontroI of Armillaria with cord-forming fungi slumps in karri (Eucalyptus diversicolor F. Mud!.) regrowth forests in southwestern Australia. New Phytologistl15, 129-138. Rayner, A. D. M. (1977a). Fungal colonization of hardwood slumps from nalural sources. I. Non-basidiomycetes. Transactions of the British Mycological Society 69, 291-302. Rayner, A. D. M. (1977 b). Fungal colonization of hardwood stumps from nalural sources. II. Basidiomycetes. Transactions of the British Mycological Society 69, 303-312. Rayner, A. D. M. (1979). Internal spread of fungi inoculated into hardwood slumps. New Phytologist 82, 505-517. Rayner, A. D. M. &: Boddy, L (1988). Fungal Decomposition of Wood -lis Biology and Ecology. John Wiley and Sons: Chichester, New Yark Brisbane, Toronto, Singapore. Rayner, A. D. M. &: Webber, 1- F. (1984). Interspecific interactions - an overview. In The Ecology and Physiology of the Fungal Mycelium (ed. D. H. Jennings &: A. D. M. Rayner), pp. 383-417, Cambridge University Press: Cambridge, U.K.
(Accepted 6 December 1994)
956 Rishbeth, J. (1976). Chemical treatment and inoculation of hardwood stumps for control of Armillaria mellea. Annals of Applied Biology 82, 57-70. Roth, L, Rolph, L &: Cooley, S. (1980). Identifying infected Ponderosa Pine slumps to reduce costs of controlling Armillaria root rot. Journal of Forestry 78, 145-151.
Schutt, P. (1985). Control of root and butt roots: limits and prospects. European Journal of Forest Pathology 15, 357-363. Shaw, C. G. III &: Kile, G. A. (1991). Armillaria Root Disease. United States Department of Agricullure, Forest Service, Agriculture Handbook No. 691, Washington D.C, U.S.A. Swift. M. J. (1970). Armillaria mel/ea (Vahl ex Fries) Kummer in central Africa: studies on substrate colonization relating to the mechanism of biological control by ring-barking. In Root Diseases and Soil-borne Pathogens (ed. T. A. Tousson, R. V. Bega &: P. E. Nelson), pp. 150--152. University of California Press: Berkeley, Los Angeles, London. Worrall, J. J. (1991). Competitive relationships between Armillaria calvescens and Tricholomopsis platyphylla. Phytopathology 81, 1141.
951
Printed in Great Britain
Effects of the cord-forming saprotrophs Hypholoma australe and Phanerochaete filamentosa and of ammonium sulphamate on establishment of Armillaria luteobubalina on stumps of Eucalyptus diversicolor
M. H. PEARCE 1 ,,., E. E. NELS OW AND N. MALAJCZUK 1 1 2
Division of Forestry, CSIRO, Private Bag, P.O. Wembley, WA., 6014, Australia USDA, Forest Service, Pacific Northwest Research Station, Forestry Sciences Laboratory, 3200 Jefferson Way, Corvallis, Oregon, 97331, U.SA.
Freshly cut karri (Eucalyptus diversicolor) thinning stumps were simultaneously inoculated with the pathogen Armillaria luteobubalina and either Hypholoma australe or Phanerochaete filamentosa. The stumps were inoculated with H. australe and P. filamentosa either above- or below-ground, and treated with either 40% (w/v) aqueous ammonium sulphamate solution (AMS) or water. Fungal colonization 2 yr after inoculation was assessed. AMS treatment had several highly significant effeds. Without AMS, neither biocontrol agent significantly reduced Armillaria colonization. However, with AMS, both P. filamentosa and H. australe significantly reduced Armillaria colonization, P. filamentosa being more effedive. Below-ground inoculation was more effedive in reducing Armillaria colonization than above-ground inoculation. In the absence of the biocontrol agents, AMS increased below-ground colonization by Armillaria compared with nonAMS treated stumps, but reduced colonization at and above ground level. Stump decay was increased, and coppice occurrence reduced, with AMS treatment. The naturally-occurring fungi Stereum hirsutum and Trametes versicolor fruited only on AMS stumps, whereas Chondrostereum purpureum fruited only on non-AMS stumps. Fruiting by Hypholoma australe was enhanced by AMS, whereas Armillaria luteobubalina fruiting was significantly greater on non-AMS stumps.
Armillaria species cause root and butt rot of many economically important forest and horticultural tree species worldwide (Shaw & Kile, 1991). Stumps are commonly colonized by the pathogen, leading to infection of nearby trees. A number of control measures have been used against Armillaria spp. However, the methods advocated most frequently, e.g. physical removal of stumps and large roots (Roth, Rolph & Cooley, 1980), and soil fumigation around infected hosts or direct injection of fumigants into infected hosts (Filip & Roth, 1977; Munnecke et a/., 1981) can be prohibitively costly in forests. Moreover, the use of fumigants toxic to other soil micro-organisms, fauna and stump decay fungi may be environmentally unacceptable (Schutt, 1985). An alternative method may be to inoculate stumps with saprotrophic wood decay fungi that either prevent or reduce Armillaria colonization (Rishbeth, 1976; Pearce & Malajczuk, 1990 a). Pearce & Malajczuk (1990a) found that although inoculation of eucalypt stumps with Trameles versicolor (L. :Fr.) Pilcit, Stereum hirsutum (Willd.: Fr.) Pers. and Xylaria hypoxylon (L.: Fr.) Grev. significantly reduced the volume of stumps colonized by Armillaria luteobubalina Watling & Kile, the test fungi were unable to colonize the subcortical region of stumps rapidly and so did not prevent establishment of Armillaria in this region. Cord-forming wood decay fungi such as Hypholoma and Phanerochaete species have a niche similar to Armillaria and share its capacity for rapid subcortical mycelial growth • Current address: 10 Goodall St, Lesmurdie, 6076, Western Australia.
(Rayner, 1977 b, 1979), and may be important in biological control (Dowson, Rayner & Boddy, 1988a, b, c; Rayner, 1977b, 1979; Pearce & Malajczuk, 1990a; Worrall, 1991). Also, chemical treatment of stumps, e.g. with ammonium sulphamate (AMS) may lead to rapid colonization of stumps by fungi which preclude entry of Armillaria (Rishbeth, 1976). This paper reports the results of a field trial in southwestern Australia to assess the efficacy of inoculating kani (Eucalyptus diversicolor F. Muel!.) thinning stumps with cordforming wood decay fungi, in combination with chemical (AMS) treatment, to control Armillaria luteobubalina biologically. A similar biocontrol study, undertaken concurrently but with antagonistic Trichoderma isolates, is reported separately (Nelson, Pearce & Malajczuk, 1995).
MATERIALS AND METHODS The experimental design included two test fungi, two stump inoculation techniques (above- and below-ground), two chemical treatments (AMS and water), and two slightly different control treatments. There were five replicates of each treatment. Fungal strains One strain each of Hypholoma australe nom. ined. (MP. 225, M. H. Pearce, and referred to in Pearce & Malajczuk, 1990a as Hypholoma sp.) and Phanerochaete filamentosa (Berk. & M. A.
952
Biocontrol of Armillaria with cord-forming fungi Curtis) Burds. (MP. 226, M. H. Pearce) were used. The P. filamentosa strain was obtained from a mycelial cord attached to a fruit body on a Banksia grandis Willd. stump, in a jarrah (Eucalyptus marginata Donn. ex Sm.) forest site at Bickley, Western Australia, in 1984. The A. luteobubalina strain is described in Pearce & Malajzcuk (1990a).
inoculated with the test fungi were (a) stumps left intact (apart from the Armillaria inoculation), and (b) non-colonized dowels were inserted into drill holes similar to the AGL fungal inoculation treatments. One replicate of each treatment was included in each of the stump size groups.
Sampling methods and assessment of colonization Study site The study site (ca 4 ha) was located in an 11-yr-old karri regrowth forest near Pine Creek ca 30 km west-south-west of Manjimup, Western Australia (lat. 34° 14/ S, long. 116° 9/ E). Additional site data are provided by Pearce & Malajczuk (1990a). A. luteobubalina was present adjacent to the site, and in the same soil type (red earth).
Preparation of inoculum Two different types of test fungus inoculum were used. In one, Ramin (Gonystylus sp.) dowelling rods (6 cm xl cm diam.) were soaked in 2 % malt extract solution for 1 h, autoclaved at 121°C for 30 min, placed onto 3% malt extract agar in 9 cm diam. Petri plates, and inoculated with two 1 em 2 agar discs of the test isolates. In the other, 6 cm x 3 em diam. stem sections from freshly felled 3-yr-old karri were autoclaved for 1 h in 2 I flasks with 100mI of distilled water. After cooling, several of the stem sections in each flask were inoculated with agar discs of the test fungi. The Armillaria inoculum was also prepared by this second method. The sealed agar plates and flasks were incubated in darkness at 20° for 5 and 8-12 wk respectively, by which time the dowels and stem sections were thoroughly permeated with mycelium.
Stump inoculations In May (late autumn) 1987, 60 suppressed but otherwise healthy karri saplings were felled and stumps (8'0-11'2 cm diam. and 30 em high) cut horizontally, using a chain saw. Stumps were identified with an aluminium or stainless steel tag and galvanized naiL and were divided equally into five groups with cut surface diameters of 8'0-8'6,8'7-9'2,9'3-9'9, 10'0-10'6 and 10'7-11'2 cm. Half of each of the size groups of stumps were poisoned with 40% wjv ammonium sulphamate (AMS) applied in two separate applications of ca 20 ml, sufficient to cover the stump surface. A major lateral root on all stumps was inoculated with A. luteobubalina by removing a section 6 cm long at the root crown and replacing it with two inoculum sections against the fresh wound and packing them in place, to simulate spread of the fungus via root contact. The inoculated root was re-covered with soil. Stumps were inoculated with a single test fungus either (1) above ground level (AGL) by inserting the colonized dowels into three 1 cm diam. holes drilled to a depth of 6 cm eqUidistant around the stump and near the base (drilled downwards at an angle of ca 45° towards the stump centre), or (2) below ground level (BGL) by placing karri stem inoculum on a cut root on the opposite side of the stump to the A. luteobubalina inoculum, follOWing the same procedure as the A. luteobubalina inoculation. Control treatments not
The presence of basidiomes and coppice regrowth on stumps was regularly monitored dUring May and June of 1988 and from March to June in 1989. In June-July 1989 the stumps were excavated and removed with as much of the root system intact as was possible (in most cases there was a single taproot with smaller lateral roots). The stumps were transported to the laboratory, debarked and sectioned transversely at 10 cm intervals in either direction from the ground line on a band saw, and later split longitudinally into halves or quarters with a chisel. The top section, 20-30 em AGL, of each stump was discarded as it was not required for analysis. The three-dimensional positions and types of decayed or discoloured zones of the remaining sections were recorded, and representative small wood chips sampled from these zones were plated onto 3 % malt extract agar (with or without 30 ppm streptomycin sulphate) and onto the Goldfarb, Nelson & Hansen (1989) medium but supplemented with 30 ppm streptomycin sulphate and 30 ppm Rose Bengal, to correlate decay patterns with the presence of various fungi (Rayner 1977 a, b, 1979; Pearce & Malajczuk, 1990a). After sampling, some of these stump sections, particularly P. filamentosa treatments, were also incubated in sealed plastic bags' for 2-3 wk (to allow for fungal growth to occur) to assist in identification of decay zones. The percentage of stump diameter colonized by A. luteobubalina was measured at each of the above-mentioned 10 em intervals after removal of the bark layer, This percentage was based on the mean of two representative perpendicular diam. measurements for each level (as per Pearce & Malajczuk, 1990a).
Approximate wood density (as a measure of decay) was determined for stump sections (10-20 em AGL, 0-10 cm AGL, 0-10 em BGL and 10-20 em BGL) by displacement of water, drying for 3 wk at 70° and weighing.
Statistical analysis Analyses of variance were carried out on the raw data for the percentage colonization and wood density values. Also, as percentage colonization by A. luteobubalina varied from 0 to 62 %, 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. Also, analyses of variance were undertaken on the AMS data alone (i.e. excluding the non-AMS data) because of the overwhelming effects that AMS treatment had on the results, i.e. to determine if there were significant fungus treatment effects in the AMS data which may have been masked in the overall data analysis.
M. H. Pearce, E. E. Nelson and N. Malajczuk
953
Chi square analyses were undertaken to examine the effects of AMS on coppice regrowth and fruiting by naturally occurring and inoculated fungi.
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Coppice presence on stumps was greatly reduced by AMS application compared with the non-AMS (water treated) controls, particularly at the end of year 1 (Table 1, P < 0'001 for 1988 and P < 0-01 for 1989). The water treated stumps with live coppice occurred equally between both the five size groups of stumps, and the different stump treatment combinations, in 1988 and 1989. Fruiting on stumps by some naturally occurring fungi was significantly affected by AMS application. Stereum hirsutum fruited only on AMS treated stumps (on 13 stumps in 1988 and 20 in 1989), whereas Chondrostereum purpureum (Pers.:Fr.) Pouzar fruited only on non-AMS treated stumps (two in 1988 and six in 1989). Trametes versicolor fruited on two AMS stumps in 1989, but not on any non-AMS treated stumps. Hypholoma australe fruiting was enhanced by AMS treatment (X 2 = 3'28, 0'05 < P < 0'10, based on 1988/89 combined data). This fungus fruited on two Hypholomainoculated AMS stumps and one non-AMS stump in 1988 and on five AMS and one untreated stump in 1989. A. luteobubalina fruited only in 1989, more so on non-AMS stumps (eight) than on AMS stumps (two) (X 2 = 4'32, P < 0'05), despite being present in portions of the bark and/or sapwood of all stumps (but refer to isolations results below). In three cases, A. luteobubalina and H. austmle fruited on the same stump, with fruiting of each fungus occurring only on opposite sides of these stumps, corresponding with their respective colonization zones. The stump excavations indicated that P. filamentosa fruited on three stumps (two AMS and one non-AMS stumps), underneath the bark. Mycelial cords of this fungus were present in the soil around these stumps.
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Stump colonization by A. luteobubaIina and other fungi The all-data analysis (AMS and non-AMS treated stumps) indicated that whilst all main effects were significant (fungus P < 0'001, chemical P < 0'01, stump section P < 0·00l). Table 1. Effect of 40% aqueous ammonium sulphamate treatment of Eucalyptus diversicoloT sapling stumps on coppice regrowth, 1 and 2 yr after treatment No. of stumps with Year observed
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June 1988
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there were two significant first order interactions (fungus x chemical P < 0'05, and chemical x stump section P < 0'001) which affected stump colonization by A. luteobubalina. There were no other significant interactions (at P < 0'05), and stump diameter had no significant effect on Armillaria colonization (at P < 0'05). With the chemical x stump section interaction, AMS Significantly (P < 0'001) enhanced BGL colonization by Armillaria compared with non-AMS stumps, whereas at GL (ground level) and AGL Armillaria colonization was reduced by AMS application (Fig. 1). With the fungus x chemical interaction, there was no significant fungus effect on Armillaria colonization with the non-AMS stumps, but there were some highly significant (P < 0'001) fungus effects with AMS treated stumps (Fig. 2). Phanerochaete filamentosa was more effective in reducing Armillaria colonization than was Hypholoma austmle, and BGL inoculation was more effective than AGL for both cordformers. The P. filamentosa-BGl inoculation combination was the only treatment which consistently greatly reduced (i.e. all but eliminated) Armillaria colonization at all stump levels measured, in comparison with all other treatments (Fig. 2). Armillaria colonization of the drill-control stumps (inoculated with non-colonized dowels) was slightly less than the control stumps left intact, particularly at 20 em BGl (P < 0'05 at 20 em BGl, Fig. 2). This may have been due to (a) accidental introduction of aerial colonists deeper into stump tissues during the drilling/dowel insertion process, and/or (b) more favourable micro-environmental conditions for growth of saprotrophs shortly after the drilling/dowel insertion process, i.e. more available oxygen deeper in the drilled stumps, compared with the intact stumps.
Biocontrol of Armillaria with cord-forming fungi
954
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Stump treatment Fig. 2. The effects of fungus treatment and stump section (at and below ground level) on colonization by Armillaria luteobubalina in Eucalyptus diversicolor sapling stumps treated with ammonium sulphamate. 0, at ground level; f:::., 10 em BGL; _, 20 cm BGL; bars, least significant difference (', P < 0'05; "', P < 0'001); p, PmmerocMete jilamentosa; H, Hypholoma australe; B, below ground inoculation; A, above ground inoculation; C control stumps left intact; CD, control stumps with non-colonized dowels. There was no colonization of stumps by A. luteobubalina at 10 or 20 em AGL.
The isolations from representative decay zones, together with visual assessments, indicated that fungal colonization was strongly influenced by ± AMS treatment. Firstly, the non-AMS treated stumps generally remained alive (particularly BGL), and for those non-AMS stumps inoculated with dowelling, colonization was strongly localized in most stumps around inoculation points. Secondly, and more specifically: (a) Chondroslereum purpureum was only isolated from nonAMS stumps (from 11/30 stumps), mostly from sections 2-4 (20 em AGL - 10 em BGL) but sometimes from sections 5-7, and occurred equally between the various stump treatments (as did all other naturally occurring fungi mentioned below). (b) Sterer-lm hirsulum occurred far more commonly, and more extensively, in AMS stumps than in non-AMS stumps (in 26/30 and 2/30 stumps respectively, and from all sections from AMS stumps, but only in sections 2-4 of non-AMS srumps). (c) Phanerochaele jilamentosa was successfully isolated from the sapwood of only one non-AMS treated stump (BGL inoculation), but from seven of the AMS stumps (five BGLand two AGL-inoculated stumps). For the five AMS BGLinoculated stumps, P. jilamentosa was isolated mostly from the external sapwood (which was extensively decayed), with the more inner sapwood colonized predominantly by S. hirsutum in four stumps and by T. versicolor in the fifth stump. (d) Hypholoma australe was re-isolated from 9/10 non-
AMS Hypholoma-inoculated stumps (from sections 2-5) and from all (10/10) AMS Hypholoma-inoculated stumps (from sections 2-7 in most cases). (e) Trameles versicolor was isolated from one non-AMS stump and from four AMS treated stumps, and occurred more extensively in the AMS stumps than in the non-AMS stumps. (f) Armillaria luteobubalina was present in the sapwood of 21/30 non-AMS stumps, and for the other nine non-AMS stumps, it had either not colonized below the bark or had only colonized small areas of the cambial region in the form of mycelial sheets and had not penetrated into the live sapwood (the latter also evidenced by the lack of any host reaction mechanisms, such as staining, in the sapwood). By contrast, A. luteobubalina was present in the sapwood of all AMS stumps. However, for the P. jilamentosa BGL-inoculated stumps, it was only present in traces as represented by small localized pockets in the outer sapwood, with the remainder of the sapwood colonized by saprotrophic decay fungi. Generally, the aerially-colonizing fungi (particularly S. hirsutum and to a lesser degree T. versicolor, and several infrequent unidentified fungi), were restricted mainly to the inner sapwood in belowground srump sections, with A. luleobubalina and/or the inoculated cord-forming fungi colonizing the outer sapwood. This was true also for most lateral root segments examined.
Stump decay Density of stump sections was significantly lower in AMStreated stumps thannon-AMS stumps (37'21 and 44'39 g cm- 3 respectively, LSD = 1'19, P < O·OOI). For the combined data analysis there was no significant main effect of fungus treatment on decay. However, the AMS-only data analysis indicated that the P. jilamenlosa and H. australe BGL-inoculated stumps were equally decayed significantly more than the Control and Drill Control stumps (P < 0'05), and that BGL inoculation resulted in greater decay than AGL inoculation for the two cord-forming fungi (P < 0'01 for P. jilamentosa and P < 0'05 for H. auslrale). Stump diam. significantly affected decay (P < 0'001 for combined data, and P < 0'05 for AMSonly data analyses respectively).
DISCUSSION This study has indicated that inoculation of stumps with cordforming wood decay fungi, combined with AMS treatment, significantly reduces stump colonization by A. luleobubalina, more so than the three non-cord-forming fungi tested by Pearce & Malajczuk (1990a). This is most likely because of the ability of H. australe and P. jilamenlosa to colonize stumps rapidly by subcortical mycelial growth, an ability not shared by the fungi tested by Pearce & Malajczuk (1990a), and hence negating Armillaria's normal positional advantage (wherein Armillaria can also rapidly colonize stumps by subcortical mycelial growth). The significantly greater effect of P. jilamentosa in reducing A. luteobubalina colonization of stumps more than H. australe is probably a reflection of P. jilamentosa's greater capacity for primary resource capture (Cooke & Rayner, 1984; Rayner & Webber, 1984). It has an intrinsic
M. H. Pearce, E. E. Nelson and N. Malajczuk ability to produce a more extensive network of mycelial cords than H. australe (Pearce & Malajczuk, 1990b; Pearce, unpublished data; and sensu Dowson et al., 1988a, b, c) with a resultant ability to colonize available domain by mycelial cords faster than H. australe. It is interesting to note, however, that in agar interactions (Pearce, 1990), H. australe (referred to therein as Hypholoma sp. A) is more combative against A. luteobubalina than is P. filamentosa. This apparent anomaly is easily explained, in that for a potential biocontrol organism to be successful. it does not need to be able to replace or kill the pathogen (although this would be desirable), but it only needs to occupy the niche and to resist replacement by the pathogen (sensu Rayner & Webber, 1984), which appears to be the situation in this study. The dramatic effect that AMS treatment had on reducing coppice regrowth and on enhancing colonization of hardwood stumps by naturally occurring fungi (as observed in this study) has been previously documented in the United Kingdom (Rishbeth, 1976; Rayner, 1977 a, b, 1979). Nevertheless, although AMS treatment quickly killed the stumps and markedly favoured above ground colonization of stumps by rapid decay fungi (5. hirsutum and to a lesser extent T. versicolor), all but eliminating Armillaria from above-ground sections of stumps, AMS enhanced below-ground colonization of stumps by A. Iuteobubalina (Fig. 1). This is probably due to Armillaria's positional advantage and ability to take advantage of the sudden (AMS-induced) decrease in host resistance to fungal infection. This positional advantage of A. luteobubalina limits the colonization of below-ground stump tissues by the aerial colonists to the more central sapwood (as observed in this study and by Pearce & Malajczuk, 1990a), thus enabling A. Iuteobubalina to continue to spread to new hosts via rootto-root contact. It is only when the fast-colonizing cordforming fungi, such as P. filamentosa and to a lesser extent H. australe, intervene that A. luteabubalina is significantly controlled, as they act by preventing A. luteobubalina from colonizing the outer sapwood. the pathogen's lifeline to a major foodbase. It is likely therefore, that a qualification to Rishbeth's 1976 study is required, in that whilst AMS application to stumps not already infected by Armillaria will probably prevent subsequent infection (because of rapid colonization of all sapwood by fungi such as 5. hirsutum and T. versicolor, and probably also by cord-forming fungi if they are present in the soil [sensu Dowson et al., 1988a, b, cD, it may not lead to adequate control if Armillaria is already present on the roots/root crown, sensu Swift's 1970 conclusions of the effects of ringbarking of trees on colonization by Armillaria. An important implication from this observation is that if AMS is to be used as a stump poison in Armillaria infected areas, then stumps will also need to be inoculated with suitable biocontrol (cord-forming) fungi, Le. an integrated control programme seems to be required for successful biocontrol of Armillaria. Otherwise, Armillaria may continue to be a significant problem in those areas. Notwithstanding the above, whilst AMS treatment significantly reduces AGL resource availability for A. luteobubalina (Fig. 1), one potential problem which may become evident is the necessity for the favoured cord-forming fungi (either
955
introduced or from natural colonization) to either resist replacement by these aerial colonizers, or if they are replaced, then these aerial colonists must in tum be able to either resist replacement by Armillaria, or replace Armillaria. Interaction studies to date indicate that S. hirsl,dum easily replaces P. filamentosa on agar, but it does not replace H. australe (M. H. Pearce, unpublished data). However, A. luteobubalina is unable to replace S. hirsutum or T. versicolor (Pearce, 1990). This study supports the views of Dowson et al. (1988a, b, c), Rayner (1977 b, 1979) and Pearce & Malajczuk (1990a), on the actual or potential biocontrol activity of cord-forming fungi against Armillaria spp. Also, an attractive attribute of using cord-forming fungi as biocontrol agents is that they can be readily established in woodland soils by direct seeding with colonized wood blocks (Dowson et al., 1988a, b, c; refer also Rayner & Boddy, 1988: 402). It is consequently envisaged that if the most efficient cord-formers are used (Le. those that quickly produce extensive cord systems in soil and subsequently rapidly colonize stump roots etc.), inoculation of individual stumps may not be required, which is most favourable from an economic viewpoint. We thank Stuart Eales, David Pearce, Robert Pearce, Tony Pearce, Dr Bernie Dell and Joanne Robinson for assistance in field operations, Carol Nelson for invaluable assistance in the laboratory, Dr Tim Grove for assistance with the statistical analyses, Dr P. K. Buchanan for verification of C. purpureum, and Dr O. K. Miller Jr for taxonomic study of Hypholoma australe. We gratefully acknowledge funding from a Reserve Bank of Australia Rural Credits Development Grant for part of this study. REFERENCES Cooke, R. C. & Rayner. A. D. M. (1984). Ecology of Saprotrophic Fungi. Longman: London. Dawson, C. G., Rayner, A. D. M. & Boddy, L. (1988a). Inoculation of mycelial cord-forming basidiomycetes into woodland soil and litter I. Initial establishment. New Phytalagist 109, 335-341. Dowson, C. G., Rayner, A. D. M. & Boddy, L. (1988 b). Inoculation of mycelial cord·forming basidiomycetes into woodland soil and litter. II. Resource capture and persistence. New Phytalagist 109, 343-349. Dowson, C. G., Rayner, A. D. M. & Boddy, L. (1988 c). The form and outcome of mycelial interactions involving cord·forming decomposer basidiomycetes in homogeneous and heterogeneous environments. New PhytaJogist 109, 423-432. Filip, G. M. & Roth, L. F. (1977). Stump injections with soil fumigants to eradicate Annillariel/a mel/ea from young-growth ponderosa pine killed by root rot. Canadian Journal of Forest Research 7, 226-231. Goldfarb, B.. Nelson, E. E. & Hansen, E. M. (1989). Trichodenna spp.:growth rates and antagonism to Phellinus weirii in vitro. Mycalogia 81, 375-381. Munnecke, D. E., Kolbezen, M.)., Wilbur, W. D. & Ohr, H. D. (I 9tH). Interactions involved in controlling Armillaria mel/ea. Plant Disease 65, 384-389.
Nelson, E. E., Pearce, M. H. & Malajczuk. N. (1995). Effects of Trichadenna spp. and ammonium sulphamate on establishment of Annil/aria luteobubalina on stumps of Eucalyptus diversicolar. Mycological Research 99, 957-962. Pearce, M. H. (1990). In vitro interactions between Annillaria luteobubalina and other wood decay fungi. Mycological Research 94, 753-761. Pearce, M. H. & Malajczuk, N. (1990a). Inoculation of karri (Eucalyptus diversicoJor F. Muell.) thinning stumps with wood decay fungi for control of Annillaria luteobubalina. Mycological Research 94, 32-37. Pearce, M. H. & Malajczuk, N. (1990b). Stump colonization by Annillaria luteobubalina and other wood decay fungi in an age series of cut-over
BiocontroI of Armillaria with cord-forming fungi slumps in karri (Eucalyptus diversicolor F. Mud!.) regrowth forests in southwestern Australia. New Phytologistl15, 129-138. Rayner, A. D. M. (1977a). Fungal colonization of hardwood slumps from nalural sources. I. Non-basidiomycetes. Transactions of the British Mycological Society 69, 291-302. Rayner, A. D. M. (1977 b). Fungal colonization of hardwood stumps from nalural sources. II. Basidiomycetes. Transactions of the British Mycological Society 69, 303-312. Rayner, A. D. M. (1979). Internal spread of fungi inoculated into hardwood slumps. New Phytologist 82, 505-517. Rayner, A. D. M. &: Boddy, L (1988). Fungal Decomposition of Wood -lis Biology and Ecology. John Wiley and Sons: Chichester, New Yark Brisbane, Toronto, Singapore. Rayner, A. D. M. &: Webber, 1- F. (1984). Interspecific interactions - an overview. In The Ecology and Physiology of the Fungal Mycelium (ed. D. H. Jennings &: A. D. M. Rayner), pp. 383-417, Cambridge University Press: Cambridge, U.K.
(Accepted 6 December 1994)
956 Rishbeth, J. (1976). Chemical treatment and inoculation of hardwood stumps for control of Armillaria mellea. Annals of Applied Biology 82, 57-70. Roth, L, Rolph, L &: Cooley, S. (1980). Identifying infected Ponderosa Pine slumps to reduce costs of controlling Armillaria root rot. Journal of Forestry 78, 145-151.
Schutt, P. (1985). Control of root and butt roots: limits and prospects. European Journal of Forest Pathology 15, 357-363. Shaw, C. G. III &: Kile, G. A. (1991). Armillaria Root Disease. United States Department of Agricullure, Forest Service, Agriculture Handbook No. 691, Washington D.C, U.S.A. Swift. M. J. (1970). Armillaria mel/ea (Vahl ex Fries) Kummer in central Africa: studies on substrate colonization relating to the mechanism of biological control by ring-barking. In Root Diseases and Soil-borne Pathogens (ed. T. A. Tousson, R. V. Bega &: P. E. Nelson), pp. 150--152. University of California Press: Berkeley, Los Angeles, London. Worrall, J. J. (1991). Competitive relationships between Armillaria calvescens and Tricholomopsis platyphylla. Phytopathology 81, 1141.