Stimulation of Armillaria ostoyae vegetative growth by tryptophol and rhizomorph produced by Zygorhynchus moelleri

Tryptophol (an indole-3-ethanol analogue which is a major secondary metabolite produced by Zygorhynchus moelleri) stimulated the growth of 13 out of 18 isolates of Armillaria ostoyae in culture. Rhizomorph production of 16 out of 18 isolates of A. ostoyae was enhanced by the presence of Z moelleri in oak branch segments. Tryptophol can be considered as a growth-promoting substance stimulating the vegetative growth of A. ostoyae in culture, and Z. moelleri as a fungus stimulating the rhizomorph formation on oak wood.     

Inhibition of Armillaria by bacteria isolated from soils of the Boreal Mixedwood Forest of Ontario

Pseudomonas fluorescens, Pseudomonas spp., Bacillus spp., Enterobacter spp., Serratia spp. and Agrobacterium radiobacter were isolated from root-free soils of the Boreal Mixedwood Forest of Ontario a, nd found to be capable of inhibiting the linear growth of Armillaria ostoyae in vitro. Isolates of P. fluorescens, Bacillus spp. and A. radiobacter were the most effective inhibitors of mycelial growth. To test the ability of the bacteria to suppress rhizomorph formation, A. gallica, which produces rhizomorphs in culture more consistently than does A. ostoyae, was used; only a small fraction of P. fluorescens and Bacillus spp. isolates were capable of preventing in vitro rhizomorph formation by A. gallica.     

Rhizomorph production and stump colonization by co‐occurring Armillaria cepistipes and Armillaria ostoyae: an experimental study

In managed spruce forests, Armillaria cepistipes and A. ostoyae are efficient stump colonizers and may compete for these resources when they co‐occur at the same site. The aim of this experiment was to quantify the mutual competitive ability of the two Armillaria species in producing rhizomorphs and in colonizing Norway spruce (Picea abies) stumps. Five isolates of A. cepistipes and two isolates of A. ostoyae were simultaneously inoculated pair‐wise into pots containing a 4‐year‐old spruce seedling. For comparison, each isolate was also inoculated alone. One year after inoculation, stumps were created by cutting down the seedlings. Six months after creation of the stumps, rhizomorph production and stump colonization were assessed. Armillaria spp. were identified from 347 rhizomorphs and 48 colonized stumps. Armillaria cepistipes dominated both as rhizomorphs in the soil and on the stumps. Nevertheless, A. ostoyae was relatively more frequent on the stumps than in the soil and A. cepistipes was relatively more frequent in the soil than on the stumps. In both species, the ability to colonize the stumps in simultaneous inoculations was significantly reduced compared with single inoculations. In respect to rhizomorph production, simultaneous co‐inoculations had a slightly stimulatory effect on A. cepistipes and no significant effect on A. ostoyae. Our study suggests a rather neutralistic co‐existence of A. cepistipes and A. ostoyae as rhizomorphs in the soil. Concerning the ability to colonize stumps, the two species experience a mutual negative effect from the interaction, probably because of interspecific competition.     

Effects of metals and pH on in vitro growth of Armillaria ostoyae and other root and butt rot fungi of red spruce

Armillaria ostoyae, Perenniporia subacida, Resinicium bicolor and Scytinostroma galactinum, root and butt rot fungi found on red spruce, Picea rubens, were tested, in vitro, for their sensitivity to metals typically found in high elevation forest soils where red spruce grows. Rhizomorph production by A. ostoyae from woody inocula in soils from red spruce stands at three elevations at each of five mountainous sites in the eastern United States was inhibited completely in the mineral soil from all elevations at all sites, and was also reduced significantly in the organic horizon from the upper two elevations at three of the sites. Inhibition was correlated with concentrations of metal ions in the soil. Growth of rhizomorphs into an agar medium containing lead and other heavy metals was inhibited for isolates of A. ostoyae from red spruce, but not for an isolate of Armillaria gallica from sugar maple; aluminium inhibited rhizomorph growth of isolates of both species. Mycelial growth of all four root and butt rot fungi was inhibited by lead, aluminium and other heavy metals depending on the solubility and concentration of metal and pH of the medium; growth inhibition was usually greater at an initial pH of 3.5 than at pH 4.5. Metal ions inhibited radial growth of Armillaria species more than that of the other three fungi. Rhizomorph growth of Armillaria was inhibited more than radial growth. Because local spread of A. ostoyae occurs frequently by means of rhizomorph growth between near roots, increases in lead, aluminium and other metals in the forest floor may contribute to this fungus' scarcity in high elevation soils and reduced incidence of infection at these sites in the eastern United States.     

Structural responses in bark to mechanical wounding and Armillaria ostoyae infection in seedlings of Pinus sylvestris

The sequence of events leading to the restoration of an intact periderm surface in the stem bark of Pinus sylvestris following wounding and challenge with Armillaria ostoyae was determined. A sub-erized impervious tissue (SIT) was produced outside the necrophylactic periderm (NP). Al-though A. ostoyae initially infected wounded bark with intracelfularly growing hyphae, these died before penetrating deeply into the bark. Later, intercellularly growing rhizomorphs, which had also developed from the inoculum, penetrated the NP and infected the functional phloem/ cambial zone. Compared to the reactions in uninfected wounded bark, the A. ostoyae infection delayed the formation of NP and reduced the frequency of cells involved in lignification and in SIT, but increased the number of lignified cell layers. The rhizomorph infection reached deeper and lignification was more pronounced in defoliated seedlings than in non-defoliated ones. The importance of structural responses in defence against A. ostoyae is discussed.     

Isolation of symbiont and associated fungi from ectomycorrhizas of Sitka spruce

Isolation of symbiont and associated fungi from ectomycorrhizas of Sitka spruce from three Irish forest plantations is described. Frequent emergence of dark sterile mycelia and Oidiodendron spp., predominantly O. maius, restricted the recovery of potential symbionts. Inhibition of the former by benomyl resulted in a sixfold increase in basidiomycetes isolated. Pure culture syntheses confirmed the ectomycorrhizal ability of potential symbiont isolates and demonstrated the parasitic nature of dark sterile mycelial types. Antagonism of Mycelium radicis atrovirens, Phytophthora cinnamomi and Heterobasidion annosum by O. maius observed in paired cultures supports the possible involvement of this species in biological control of conifer root pathogens.     

Stump infection by Armillaria in first-rotation conifers

Information about the entry of Armillaria into first-rotation pine and spruce stands was obtained by searching for infected stumps, rhizomorph systems or trees that had been killed. In pines Armillaria foci were very rare. In pure Norway spruce Armillaria lutea and A. mellea were detected in stumps but rhizomorphs did not extend into the soil; in Norway spruce mixed with oak, by contrast, A. lutea sometimes produced extensive rhizomorph systems. In Sitka spruce small groups of trees had been killed by A. ostoyae. All foci investigated in conifers contained different genotypes of Armillaria and probably originated from spore infection of stumps created by thinning. Some implications of these findings are discussed.     

Comparison of the virulence of Armillaria cepistipes and Armillaria ostoyae on four Norway spruce provenances

The basidiomycetes Armillaria cepistipes and Armillaria ostoyae frequently occur in the same forest stand. In this study, we determined the virulence of 20 isolates of A. cepistipes and 16 isolates of A. ostoyae on four different provenances of 2‐year‐old Norway spruce (Picea abies). Within 30 months after inoculation, 1.1 and 19.1% of the seedlings inoculated with A. cepistipes and A. ostoyae, respectively, had died or were dying. The incidence of dead and dying seedlings varied between 3 and 49% among the A. ostoyae isolates. The virulence of an isolate was positively correlated to its ability to produce rhizomorphs. One Norway spruce provenance showed significantly lower susceptibility to A. ostoyae than the other three. Rhizomorphs of both Armillaria species were attached to the root surface. The attached rhizomorphs of A. ostoyae, however, were associated with significantly more lesions. The virulence of the isolates was not correlated with their wood‐degrading capability for either of the Armillaria species.     

The influence of carbon nutrition on Armillaria ostoyae growth and phenolic degradation

Concentrations of 1, 5 and 25 g C L^?1 as glucose, fructose and sucrose were added to basal media containing 0 or 1.0 mM catechol orwa-hydroxybenzoic acid. Media were poured in petri dishes and inoculated with one of three isolates of Armillaria ostoyae(Romagn.) Herink. Armillaria ostoyae isolates usually had greater colony size and biomass yield when glucose was the carbon source as compared to fructose or sucrose. When compared to A. ostoyae growth on basal media alone, 1.0 mM catechol and /wvi-hydroxybenzoic acia added to basal media inhibited A. ostoyae growth. In a second experiment, catechol andpara-hydroxybenzoic acid degradation by three A. ostoyae isolates growing on 1, 5 and 25 g C L^?1 glucose, fructose or sucrose were measured radiometrically. Carbon source and concentration had no effect on the degradation of pzra-hydroxybenzoic acid or catechol by Armillaria ostoyae. Results of these experiments suggests that A. ostoyae can grow faster when at high carbon concentration but cannot more effectively degrade catechol or para-hydroxybenzoic acid.     

Molecular‐based identification and phylogeny of Armillaria species from Serbia and Montenegro

Armillaria causes problems of root rot, kill trees and decay wood in the forests of Serbia and Montenegro, but the species involved have not hitherto been identified. The aim of this study was to identify field isolates collected on 25 localities. Identification was based on restriction fragment length polymorphism (RFLP) analysis of intergenic spacer 1 (IGS1) region and comparisons of IGS1 sequence with those available on NCBI database. Phylogenetic analysis was performed on sequence information from selected isolates to determine possible interrelationships between isolates with different banding patterns and previously identified tester isolates of five European Armillaria species. Five Armillaria species were identified in 90 isolates obtained from forests in Serbia and Montenegro. Armillaria gallica was most frequently isolated, followed by A. cepistipes, A. mellea, A. ostoyae and A. tabescens; two isolates remained unidentified. Restriction digestion of IGS1 amplification products with AluI produced 10 RFLP patterns. Patterns G4 (400, 250, 180) for A. gallica and pattern X (400, 180, 140) for isolates 74 and 79 are reported for the first time in European isolates. Eight RFLP patterns were observed after restriction with TaqI. Two patterns each were observed for A. ostoyae and A. gallica, and one each for A. cepistipes, A. mellea, A. tabescens and isolates 74 and 79. Parsimony analyses based on the IGS1 region placed the isolates into four clades: one including A. mellea, the second containing A. gallica–A. cepistipes isolates, while isolates of A. ostoyae and A. borealis were in the third clade. Armillaria tabescens differed from all annulate species. Phylogenetic analysis supported the conclusion that European Armillaria species are closely related and separated from a common ancestor in the near past. According to this survey five European Armillaria species are present in the forests of Serbia and Montenegro, while A. borealis is not present in the studied ecosystems.     

Mycorrhizal and associated fungi of Sitka spruce in Irish forest mixed stands

Identity of mycorrhizas and isolation of symbionts and associated fungi from Sitka spruce growing in pure and mixed stands with either Japanese larch or Lodgepole pine are described and compared. More mycorrhizal types and sporocarps of the Agaricales were collected from mixed stands. Mycorrhizas of Lactarius rufus, Paxillus involutus and Suillus spp. were more prevalent on roots from mixed stands. The most common unidentified mycorrhizal type (type B) had features similar to synthesized mycorrhizas of two Basidiomycete isolates. Suillus grevillei and three un-identified types were associated specifically with Japanese larch. The main associating fungi were Oidiodendron sp. and Mycelium radicis atrovirens. The association of a “nurse” tree with Sitka spruce provides a more diverse mycorrhizal flora, the majority of which are shared between the tree species.     

Identification and partial characterization of an extracellular manganese-dependent peroxidase in Armillaria ostoyae and Armillaria mellea

Laccase and manganese-dependent peroxidase (Mn peroxidase) activities were detected in the culture media of Armillaria ostoyae and A. mellea. Mn peroxidase was produced in significantly higher quantity by the A. ostoyae isolates and was purified by chromatography from one isolate of this species. Some properties of the purified enzyme were examined (absorption spectrum, H₂O₂ and MnSO₄ optimal concentrations, pH optimum and lactate stimulation). Enzymes of potential importance in the lignin degradation (especially Mn peroxidase) by Armillaria sp. are compared to those of other root-rotting fungi. The possible role of Mn peroxidase in modulating the pathogenicity of Armillaria sp. is discussed.     

Identification of the genus Armillaria by specific amplification of an rDNA-ITS fragment and evaluation of genetic variation within A. ostoyae by rDNA-RFLP and RAPD analysis

Genetic variation among Armillaria ostoyae isolates was studied by rDNA-restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. A total of 20 A. ostoyae isolates, mainly obtained from Picea spp. of different geographical origins, were examined. Southern hybridization of whole-cell DNAs digested with AvaII and probed with biotin-labelled cloned rDNA from Saccharomyces carlsbergensis allowed the differentiation of five RFLP groups. UPGMA cluster analysis of RAPD profiles (138 scorable bands) generated by 10 decamer primers (OPA 01-OPA 10) grouped the isolates in subclusters at similarity levels between 40% and 96%, indicating high intraspecific genetic variability. Some isolates of different geographical origins subgrouped together, suggesting that similar mutational events have occurred independently and that genetic exchange and recombination occurs among the DNAs in natural populations. The potential role of historical and current spread of spruce plants on the genetic variation of A. ostoyae isolates in Europe is discussed. Using the primer pair ARM-1 and ARM-2, an Armillaria-specific ITS-DNA fragment of about 660 bp was obtained. No intraspecific RFLP of this amplicon could be revealed, indicating low genetic variability of this region. The established informative RFLP and RAPD markers and also the Armillaria-specific ITS-DNA fragment may be powerful tools for further epidemiological, phylogenetic and host-pathogen interaction studies with A. ostoyae.     

Pathogenicity and virulence of Armillaria sinapina and host response to infection in Douglas‐fir,western hemlock and western redcedar in the southern Interior of British Columbia

The frequency of infection, lesion characteristics and anatomical changes in phloem and cambial tissues caused by Armillaria sinapina were studied on inoculated trees of Douglas‐fir, western hemlock and western redcedar and compared with results of A. ostoyae inoculations previously reported on the same host species. Similar percentages of inoculations resulting in infection of roots on the three hosts indicate that A. sinapina and A. ostoyae are equally pathogenic. Armillaria ostoyae was more virulent than A. sinapina as demonstrated by fungal exudates from A. ostoyae inoculum blocks, which appeared to cause lesions on roots; the higher frequency at which lignified impervious tissue (IT) and necrophylactic periderm (NP) developed in bark and following cambial invasion, compartmentalization; the large proportion of roots that showed no visible host response; the large zones of IT formed under continuous stimulation by A. ostoyae advancing in inner bark; and the high frequency of breaching of NP barriers. Spread of A. sinapina mycelium in host species appeared slower than that of A. ostoyae, particularly in Douglas‐fir and western hemlock. In western redcedar, A. ostoyae induced stronger host responses than those following invasion by A. sinapina, which included further expansion of the induced rhytidome response, traumatic phloem resin duct formation and higher numbers of polyphenolic parenchyma comprising its barrier zone. Where damage by A. sinapina ensued, it was always associated with high inoculum potential. The ecology of virulent and less virulent species of Armillaria in natural forests is discussed.     

Mycelial fan formation of three sympatric Armillaria species on excised stem segments of Picea abies

Mycelial fan formation was studied in five Armillaria cepistipes, ten A. borealis and ten diploid and six haploid A. ostoyae strains on excised stem segments of Picea abies. Stem segments were either non‐autoclaved or autoclaved, representing dying and dead wood, respectively. To confirm the identity of mycelial fans on non‐autoclaved stem segments, re‐isolations were made and isolates characterized with microsatellite markers. Mycelial fan formation on autoclaved stem segments was fast and reliable for most of the tested Armillaria strains. On non‐autoclaved stem segments, mycelial fan formation was slower, more erratic and less predictable. Mycelial fan formation was fastest in A. cepistipes closely followed by A. borealis and was slowest in A. ostoyae. For two A. cepistipes and four A. ostoyae strains (all diploid), growth rates of mycelial fans were estimated in a time course experiment. They ranged between 5.1 and 8.7 mm/day for autoclaved and between 1.4 and 4.7 mm/day for non‐autoclaved stem segments. The haploid A. ostoyae strains also formed mycelial fans on autoclaved stem segments, but typically slower and less reliably than the diploid strains. Whether haploid strains are able to produce mycelial fans on non‐autoclaved stem segments remains unknown because of accidental diploidization of the original haploid strains which was likely caused by basidiospores introduced into the study system on the non‐autoclaved stems. Overall, the method developed in this study may be useful for further investigations into the genetic, physiological and biochemical nature of mycelial fan formation in the genus Armillaria.     

Identification of Kenyan Armillaria isolates by cultural morphology,intersterility tests and analysis of isozyme profiles

Three groups of morphologically distinct isolates were observed among nine Kenyan Armillaria isolates based on their mycelium and rhizomorphs characteristics. Seven of the isolates were interfertile with testers of North American biological species III, VII and IX. However, tests with benomyl segregants BEN 433, BEN 157-10 and BEN AVK were intersterile with the same testers and also with the European A. mellea, A. lutea and A. ostoyae. The analysis of isozyme profiles showed that morphologically similar isolates had similar isozyme profiles of esterases. Their profiles however differed from those of the European A. mellea, A. lutea and A. ostoyae. On the basis of intersterility tests and analysis of isozyme profiles, the Kenyan isolates should be considered different.     

Phylogeographic patterns of Armillariaostoyae in the western United States

Nuclear ribosomal DNA regions (i.e. large subunit, internal transcribed spacer, 5.8S and intergenic spacer) were sequenced using a direct‐polymerase chain reaction method from Armillaria ostoyae genets collected from the western USA. Many of the A. ostoyae genets contained heterogeneity among rDNA repeats, indicating intragenomic variation and likely intraspecific hybridization. Intragenomic variation was verified by visually editing base‐sequence offsets in regions with insertions/deletions, and using sequence‐specific internal primers to resequence heterogeneous regions. Phylogenetic analyses with Bayesian Inference methods were used to define groups within A. ostoyae. Analysis of A. ostoyae from outside the western USA indicated the presence of a Circumboreal group of A. ostoyae that also occurs in Utah; two other phylogeographic groups were associated with the Rocky Mountain and Pacific Northwest regions of the USA. Mixed sequence types, an indication of intraspecific hybrids, were common in some geographic regions. Hybridization events may have influenced species evolution, contributing to variation in pathogenicity and virulence. The occurrence of these groups and intraspecific hybrids also indicates that paleogeography and paleoclimate may have influenced the phylogeography of A. ostoyae. In addition, other Armillaria species were examined for evolutionary relationships with the groups of A. ostoyae. These findings will provide a basis for future research relating ecological function to genetic diversity within A. ostoyae.     

Laccase isoenzyme patterns of European Armillaria species from culture filtrates and infected woody plant tissues

Polyacrylamide isoelectric focusing with specific staining for laccase activity was used to characterize laccase from European Armillaria species (Armillaria ostoyae, Armillaria mellea, Armillaria gallica, Armillaria cepistipes). The enzyme was extracted from culture media either supplemented, or not, with pine sawdust, and also from Pinus pinaster naturally infected by A. ostoyae, or artificially inoculated with A. mellea and A. ostoyae. Some differences in banding patterns were found for Armillana isolates according to the species and the culture media, but a common band at pI = 3.4 was found in all the extracts tested, independently of their origin (culture filtrate or wood).     

Variation in virulence among isolates of Armillaria ostoyae

The virulence of Armillaria ostoyae isolates from coastal (16) and interior (33) British Columbia, elsewhere in North America (eight) and Europe (six) was assessed on 2‐year‐old Douglas‐fir seedlings in pots during a 3‐year trial. Isolates from most geographical locations infected similar proportions of seedlings, had similar average damage scores and killed a similar percentage of diseased seedlings. Isolates from the coastal region had a significantly higher probability than interior isolates that a diseased seedling received a damage score > 3 on a 1–5 scale, and coastal isolates killed a higher proportion of diseased seedlings than interior isolates. The mean damage score for isolates that had been in culture for 20–25 years was about 25% lower than that for recently collected isolates. The results indicate that the higher incidence and longer duration of mortality in the southern interior of British Columbia compared to the coast can not be attributed to greater virulence of interior isolates of A. ostoyae.     

Clones of Armillaria ostoyae and the pattern of infected juvenile lodgepole pine in Alberta,Canada

Small patches of dead trees were common among 7- to 15-year-old Pinns contorta var. latifolia in Alberta, Canada. The sizes of clones of Armillaria ostoyae, determined by pairing diploid isolates, was larger than these patches. Pairing diploid isolates on strips of Betula papyrifera provided a clear indication of clonal relationships.