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.     

Penetration of Picea sitchensis root bark by Armillaria mellea,Armillaria ostoyae and Heterobasidion annosum

Penetration of root bark tissues of Picea sitchensis by Armillaria ostoyae, Armillaria mellea and Heterobasidion annosum was examined in the absence of wounds, in superficial wounds (rhytidome tissues removed to expose the secondary phloem) and in wounds to the depth of the vascular cambium (deep wounding). Both species of Armillaria penetrated bark without prior wounding, but neither species formed rhizomorphs in this treatment. Armillaria ostoyae penetrated to 39 cell layers in depth by 48 days after inoculation of unwounded bark, whereas A. mellea penetrated 25 cell layers in the same time. Armillaria mellea penetrated superficial wounds significantly more rapidly than did A. ostoyae. Both species produced rhizomorphs within wounded host tissues. Inoculation of deep wounds with Armillaria resulted in a greater depth of bark necrosis with A. mellea than with A. ostoyae. In the absence of wounding, H. annosum failed to penetrate root bark tissues, but in both superficial and deep wounds hyphae penetrated beyond the ligno–suberized boundary zone (LSZ) by 12 days after inoculation. Where no inoculations were made, superficial or deep wounding led within 25 days to the restoration of a structurally continuous LSZ, and by day 48 the wound periderm (WP) was fully differentiated. In inoculated wounds, however, formation of the LSZ and WP was delayed or inhibited in most trees, particularly following inoculation with A. ostoyae or A. mellea. Suberization in the LSZ and WP remained diffuse and discontinuous 48 days after inoculation. Moreover, the presence of WP did not prevent further penetration of the tissues by the pathogens. Variations between trees in the depth of pathogen penetration were noted, possibly indicating differing susceptibilities of individual host genotypes. The possible host factors involved in resistance to penetration of root bark tissues by Armillaria and Heterobasidion are discussed.     

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.     

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.     

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.     

Tree vigour and the susceptibility of Douglas fir to Armillaria root disease

In this study the effects of thinning, fertilization and pruning on the vigour of Douglas fir (Pseudotsuga menziesii) and its susceptibility to Armillaria root disease were explored. Tree vigour was defined as the relative capacity for tree growth, expressed as the above-ground biomass increment per unit of photosynthetic tissue, or growth efficiency (GE). It has been hypothesized that trees with higher GE can better resist pathogen attack, and that GE can be used as a predictor of tree susceptibility to disease. In a previous study, four Douglas fir plantations were thinned, fertilized and pruned in all combinations, and the effects of these treatments on tree vigour were measured after 10 years. Root disease was not a factor in the initial study design, and mortality was ignored until 8 years after the treatments were applied. The results of an earlier study were utilized and the correlation between Armillaria root disease incidence and the effects of earlier stand treatments on tree growth was tested. Armillaria ostoyae was the primary mortality agent in the study area. Disease incidence on infested subplots ranged from 2 to 20%. Armillaria ostoyae incidence was the highest at medium tree density (6.1%), slightly lower on the low density (5.6%), and lowest on the unthinned plots (3.8%). There were no significant correlations between disease incidence and previous tree growth. The vigour of trees that became symptomatic or died by 1993 was not significantly different in 1983–85 from the vigour of trees that remained asymptomatic. On these sites, in areas of infestation, A. ostoyae is killing the largest, fastest growing trees, as well as less vigorous trees. Armillaria continues to cause mortality, regardless of the growth efficiency or growth rate of the host.     

Identification of Armillaria species in Japan using PCR-RFLP analysis of rDNA intergenic spacer region and comparisons of Armillaria species in the world

Armillaria species from Japan were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the intergenic spacer region-1 (IGS-1) of ribosomal DNA (rDNA). Eleven different digestion patterns by restriction endonuclease Alu I were found among 70 isolates of seven Armillaria species in Japan. Isolates within Armillaria nabsnona, A. ostoyae, A. cepistipes, and Japanese biological species E showed the same Alu I digestion patterns. Five Alu I patterns were detected for A. gallica, three patterns for A. mellea, and two patterns for A. tabescens. Seven Armillaria species in Japan were clearly distinguished by using the profiles obtained when PCR products were digested with Alu I, Msp I, and Hae III restriction enzymes. There was considerable variability of Alu I restriction sites within the IGS-1 between the isolates of five Armillaria species, A. gallica, A. nabsnona, A. cepistipes, A. mellea, and A. tabescens, in Japan and those of their European and North American counterparts.     

Signs and symptoms associated with Heterobasidion annosum and Armillaria ostoyae infection in dead and dying mountain pine (Pinus mugo ssp. uncinata)

Symptoms and signs associated with root rot caused by Heterobasidion annosum or Armillaria ostoyae in mountain pines (Pinus mugo ssp. uncinata) were investigated in the Swiss Alps. A sample of dying or recently dead mountain pine trees (≥12 cm d.b.h.) and saplings (<1.3 m height) was assessed for root pathogen infection by taking root samples followed by isolations in the laboratory. From a subsample, an additional core was taken from the butt of each tree and evaluated in the same fashion. A total of 157 dying or recently dead mountain pine trees and 184 saplings with roots infected by either of the two pathogens or which lacked infection were analyzed using logistic regression models. The main objectives were to determine the most prominent symptoms induced by the fungi (resinosis), signs of the fungi (mycelia, fruiting bodies and rhizomorphs), and tree characteristics (d.b.h./height and evidence of wounds) that would allow an easy and reliable determination of H. annosum and/or A. ostoyae infection of mountain pines in the field. Heterobasidion annosum caused both root and butt rot on mountain pine, whereas A. ostoyae was mostly restricted to the root systems of the trees sampled. The most discriminating sign for the presence of A. ostoyae infection was the presence of characteristic mycelial fans, and for H. annosum root rot the presence of H. annosum mycelia (sheets of paper‐thin mycelium and mycelial pustules). In addition, resinosis was a powerful predictor for A. ostoyae in trees. Symptoms and signs indicating A. ostoyae or H. annosum infections were more reliable for saplings than for mature trees. Armillaria rhizomorphs were not useful in detecting A. ostoyae infection and, if present, were often formed by saprophytic Armillaria species. Heterobasidion annosum fruiting bodies were rarely observed and poorly reflected the widespread occurrence of this pathogen in the mountain pine forests.     

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).     

Ecology and distribution of Armillaria species in Norway

The occurence of Armillaria species was assessed in Norway, enabling the northern‐most distribution of this genus to be determined in Europe. Four Armillaria species were found in Norway. Armillaria borealis was the most common species occurring on woody vegetation to the permafrost zone (ca. 69°N). Armillaria cepistipes was present in southern and central Norway, but was not found further than 66°N. Armillaria solidipes and Armillaria gallica were rare, found at only one locality each; 59°40′ and 59°32′, respectively. Armillaria species were found on 14 hosts, but there was no significant difference between occurrence of A. borealis and A. cepistipes on declining and dead trees. Phylogenetic analyses separated each species into separate clades. All isolates of A. borealis, except one, and most isolates of A. solidipes were in separate clades. However, a subclade within the A. borealis clade was formed of two A. ostoyae and one A. borealis isolates. Two small A. cepistipes genets were found in a declining oak stand.     

Geographical distribution of the Armillaria species in The Netherlands in relation to soil type and hosts

The geographical distribution of the annulate Armillaria species was studied in The Netherlands during the period 1983–1992. Armillaria gallica (incl. A. cepistipes), A. mellea and A. ostoyae appear to be widespread, the first two species being rather common on broad-leaved hosts growing on clay and loess soils, and the third species common on both broad-leaved and coniferous hosts on acid sandy soils. The distribution of the Armillaria species encountered was primarily determined by soil type. From a silvicultural point of view, A. ostoyae is the most important species, being pathogenic and occurring most frequently on sandy soils, the soil type predominantly used for forestry in The Netherlands.     

Ecology of Armillaria species on conifers in Japan

Distribution, host preference and pathogenicity of Japanese Armillaria species on conifers were investigated on the basis of field collections of 65 isolates. We identified seven Armillaria species from 19 conifer species including six major Japanese plantation conifers using mating tests and sequences of the translation elongation‐1 α gene. Armillaria mellea, Armillaria ostoyae, Armillaria cepistipes and Armillaria sinapina were frequently collected, whereas Armillaria nabsnona, Armillaria tabescens and a biological species Nagasawa’s E were rare. On the basis of host condition when the isolates were collected, A. mellea, A. ostoyae, A. cepistipes and A. tabescens are considered as moderate to aggressive pathogens of conifers in Japan.     

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.     

Parasitic association of the mycetophagous wood nematode,Bursaphelenchus fraudulentus with the honey fungus Armillaria ostoyae

Honey fungi, Armillaria spp. are common and economically important pathogens of a wide range of tree species grown both in the forest and orchard cultures worldwide. Our field research in 73 forest stands across Poland has shown a common association of the mycetophagous wood nematode, Bursaphelenchus fraudulentus with Armillaria‐infected trees. The data reported here provide the first insight into the parasitic interaction of a nematode and Armillaria ostoyae. In laboratory conditions, B. fraudulentus reproduced on A. ostoyae, caused substantial damage to its mycelium and, finally, killed the whole colony within a short time. In ageing cultures, the nematode produced resting (dauer) juveniles. After artificial inoculation to Armillaria‐infected trees, the nematode population persisted under the bark, continued feeding and mass reproduction on the mycelium, and dispersed over the mycelial fans. The ability of B. fraudulentus to develop and reproduce on the surface and inside the fungal rhizomorphs suggests that these structures could facilitate the nematode dispersion in the environment. Since B. fraudulentus is not pathogenic to the host tree, its parasitic association with A. ostoyae may contribute to natural regulation of this fungus in the environment. The observed characteristics of this nematode suggest that it could potentially be used as a biocontrol agent of honey fungi in forest and orchard trees.     

Armillaria in an ancient broadleaved woodland

In an ancient broadleaved woodland in eastern England that had been coppiced regularly for over 700 years Armillaria mellea and A. gallica were common, A. tabescens was moderately frequent, whilst A ostoyae and A cepistipes were local. Fruit bodies were often abundant in areas coppiced the previous winter. The largest focus of A gallica occupied 9 ha and was probably over 500 years old. Large trees were rarely killed but A mellea caused some root decay in birches. The number of coppice shoots killed by Armillaria was small but became greater as the period since coppicing increased; A gallica was most often involved. Alder coppice was the most, and hazel coppice the least, affected. The productivity of coppiced woodland in general seemed little reduced by Armilaria.     

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.     

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.     

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.     

Ecology of Armillaria species on silver fir (Abies alba) in the Spanish Pyrenees

? We describe the distribution and the ecology of three Armillaria species observed in silver fir (Abies alba) forests of the Pyrenees.

? We surveyed the presence and abundance of Armillaria above and belowground in 29 stands. Isolates were identified by the PCR-RFLP pattern of the IGS-1 region of their ribosomal DNA. We measured several ecological and management parameters of each stand in order to describe Armillaria infected sites.

? Armillaria cepistipes was the most abundant of three species observed. Armillaria gallica was dominant in soils with a higher pH and at lower elevations. Armillaria ostoyae seemed to be more frequent in stands where A. alba recently increased its dominance relative to other forest tree species. Thinning activities correlated with an increased abundance of Armillaria belowground. In 83% of the stands the same Armillaria species was observed above and belowground.

? It seems that in a conifer forest, A. cepistipes can be more frequent than A. ostoyae, a virulent conifer pathogen. Since logging is related to a higher abundance of Armillaria in the soil, the particular Armillaria species present in a given stand could be considered an additional site factor when making management decisions.

     

Characterization of North American Armillaria species: genetic relationships determined by ribosomal DNA sequences and AFLP markers

Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS‐1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data, the nLSU region is less variable among Armillaria species than the ITS + 5.8S and IGS‐1 regions (nLSU < ITS + 5.8S < IGS‐1). Phylogenetic analyses of the rDNA sequences suggested Armillaria mellea, A. tabescens and A. nabsnona are well separated from the remaining Armillaria species (A. ostoyae, A. gemina, A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes). Several Armillaria species (A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes) clustered together based on rDNA sequencing data. Based on the isolates used in this study, it appears that techniques based on IGS‐1, ITS + 5.8S, and/or D‐domain/3′ ends of nLSU are not reliable for distinguishing A. calvescens, A. sinapina, A. gallica and A. cepistipes. However, AFLP data provided delineation among these species, and AFLP analysis supported taxonomic classification established by conventional methods (morphology and interfertility tests). Our results indicate that AFLP genetic markers offer potential for distinguishing currently recognized North American Biological Species (NABS) of Armillaria in future biological, ecological and taxonomic studies.