Armillaria ostoyae in managed coniferous forests in Kastamonu in Turkey

Although several Armillaria species have been reported in Turkey, there is little information about their ecology in Turkish forests. In this study, we investigated five forest stands, approximately 5–74 ha in size, in Kastamonu province in the Black Sea Region of Turkey for the presence of Armillaria species in stumps and logs. The stands were mixed Abies nordmanniana ssp. bornmülleriana and Pinus sylvestris forests managed using a selective cuttings system; the proportion of fir in the total number of stems and stumps ranged from 36 to 98%. Based on sequence analysis of the internal transcribed spacer and intergenic spacer regions of the rDNA, all rhizomorphs sampled from the stumps and logs were of Armillaria ostoyae. The size of the genets was estimated with random amplified microsatellites analysis of the isolates and ranged from single stumps to approximately 450 m². One to seven genets were found in each stand. These results indicate that the genets had arisen from spores and vegetative spread was limited on most sites.     

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.     

Reaction of Armillaria ostoyae to forest soil microfungi

Fungi isolated from the oak (Quercus robur) rhizosphere were tested for their effects on rhizomorph formation and growth of 16 isolates of Armillaria ostoyae sampled in three localities in western Poland. The number of rhizomorphs, number of rhizomorph apices, and rhizomorph length and weight increased most in the presence of Penicillium lanosum, Penicillium notatum, Cylindrocarpon destructans, Penicillium spinulosum and Mycelium radicis atrovirens α and, to a lesser extent, in the presence of Nectria grammicospora. Inhibition of rhizomorph formation was caused by Trichoderma hamatum and Trichoderma viride in two A. ostoyae isolates and by M. radicis atrovirens α and P. spinulosum in one A. ostoyae isolate. It is suggested that variation in sensitivity to microbial stimulation within A. ostoyae is associated with the environmental and nutritional conditions of its original habitat. Isolates from nutrition‐rich localities, with 20% of the land area covered by deciduous trees, were particularly susceptible to stimulation by rhizosphere fungi.     

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.     

Development of species‐specific PCR primers on rDNA for the identification of European Armillaria species

Attempts to design species‐specific PCR primers from six European Armillaria species in the ribosomal RNA genes are reported. Primers were developed on the basis of the nucleotide sequence variability of the internal transcribed spacers (ITS) and the intergenic spacer (IGS1) of the ribosomal DNA. Four sets of primers gave specific PCR products for Armillaria tabescens, Armillaria mellea and Armillaria ostoyae. However, due to the high sequence similarities between Armillaria borealis and Armillaria ostoyae and between Armillaria cepistipes and Armillaria gallica no species specific amplification was obtained for these taxa.     

Effects of wounding and fungal infection with Armillaria ostoyae in three conifer species. II. Host response to the pathogen

Structural responses in the bark and wood were described following penetration by Armillaria ostoyae in the roots of 20‐ to 30‐year‐old Douglas‐fir, western hemlock and western redcedar trees. Tissue necrosis presumably caused by fungal exudates was commonly observed at inoculum contact. In Douglas‐fir and western hemlock, A. ostoyae interfered with the initiation of active defence mechanisms involving the development of a lignified zone of impervious tissue (IT), necrophylactic periderm (NP) formation and compartmentalization of infected woody tissue. Breaching of IT and NP barriers was frequent, particularly around the clusters of sclereid cells in western hemlock. In western redcedar, the IT zone was inconspicuous. Induced rhytidome formation occurred in western redcedar either simultaneously with or after completion of NP development. The formation of this tissue facilitated en masse sloughing of infected tissue from the surface of roots. In western redcedar, traumatic phloem resin ducts formed in tangential bands surrounding the margin of expanded lesions. Effective compartmentalization in western redcedar was achieved by a barrier zone comprised of a higher‐than‐average number of axial parenchyma that accumulated polyphenolic deposits. A combination of host‐mediated defence mechanisms in western redcedar resulted in a significantly higher frequency of effective resistance reactions than in western hemlock or Douglas‐fir.     

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.     

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.     

Lesion formation and host response to infection by Armillaria ostoyae in the roots of western larch and Douglas‐fir

The process of lesion formation and host response to natural infection by Armillaria ostoyae were studied in the roots of western larch (Larix occidentalis) and Douglas‐fir (Pseudotsuga menziesii ssp. glauca) trees in the three age classes, 6–8, 18–19 and 85–95 years. The characteristics of lesions on infected roots were recorded and bark samples were dissected from infection points and lesion margins in the field and stored in liquid nitrogen for macroscopic study in the laboratory. Infection in the roots of 6‐ to 8‐year‐old trees advanced freely, overcoming any host resistance, quickly girdling the root collar and killing the trees. In 18‐ and 19‐year‐old trees, however, 43% of infections on western larch and 27% of the infections on Douglas‐fir roots were confined to lesions bounded by necrophylactic periderms with multiple bands of phellem. Host response was similar in 85‐ to 95‐year‐old trees, but the percentage of confined lesions was higher than in younger trees. The results suggest that larch shows resistance to A. ostoyae at a younger age and with greater frequency than Douglas‐fir.     

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.     

Armillaria ostoyae associated with dying 60‐year‐old Scots pines in northern Turkey

We investigated an Armillaria root disease centre in a 60‐year‐old, naturally regenerated Pinus sylvestris stand located at 1250 m altitude in Sinop province in northern Turkey. Based on sequence analysis of the internal transcribed spacer region of the rDNA, the mycelial fans sampled from the infected trees were Armillaria ostoyae. Multilocus genotyping indicated that the fans belonged to a single genet, at least 0.2 ha in size. Drought as a predisposing factor is discussed. The study area received 50–70 and 25–50% of the normal precipitation in May, June and August in 2009 and in August and September in 2010, respectively. Moreover, earlier excavation work to prepare a forest road had probably disrupted the water table in the affected stand.     

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.     

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.     

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.     

Spatial distribution and persistence of Heterobasidion parviporum genets on a Norway spruce site

Spatial distribution of Heterobasidion genets over a period of ca 50 years in two successive generations of Norway spruce (Picea abies) was unravelled. The genets were first identified in 1993 in a naturally regenerated 43‐year‐old spruce stand that had been thinned the previous winter. Heterobasidion parviporum was found in 17.5% of the old stumps of the previous spruce generation. Nine genets were identified on the study plot; seven of them were present in old stumps of the previous tree generation and two only in the new spruce generation. Eighteen spruce trees of the new generation were infected, 15 of them by vegetative growth of genets originating from the old stumps. The study plot was investigated again in 2005. No new genets had been established after thinning, and three old genets had died out. The remaining genets had infected five new trees, most likely from the thinning stumps of diseased trees. At the age of 56 years, 16.1% of the residual spruces were infected by Heterobasidion. The results of this study suggest that if spore infection to stumps of spruce can be prevented, the decay frequency caused by H. parviporum will not necessarily increase in successive generations.     

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.     

Necrophylactic periderm formation in the roots of western larch and Douglas‐fir trees infected with Armillaria ostoyae. II. The response to the pathogen

Periderm formation was studied in bark samples collected from the roots of western larch (Larix occidentalis) and Douglas‐fir (Pseudotsuga menziesii) trees infected with Armillaria ostoyae. Necrophylactic periderms were formed in advance of infection and successfully restricted continued fungal spread in 68 and 45% of the samples collected from 10‐ and 27‐year‐old western larch, respectively. However, all periderms formed in 11‐ and 25‐year‐old Douglas‐fir had been breached by the advancing fungus. In both species, necrophylactic periderms were commonly breached at the junction of the periderm with the vascular cambium. In western larch, stone phellem often comprised the external phellem layer of necrophylactic periderms with multiple bands of phellem. In 27‐year‐old western larch, infection was often confined to discrete lesions bounded by multiple periderms with multiple bands of phellem. In both tree species, phellem production was greater in response to infection than in response to abiotic wounding.     

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.     

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.     

Epidemiology of Armillaria root disease in Douglas‐fir plantations in the cedar‐hemlock zone of the southern interior of British Columbia

Results are presented from several studies on the epidemiology of Armillaria ostoyae in Douglas‐fir plantations in the interior cedar‐hemlock (ICH) biogeoclimatic zone of British Columbia. Two plantations were monitored for mortality by A. ostoyae and other agents for 35 years after establishment. In these and other plantations ranging in age from 7 to 32 years, one or more of the following factors were determined: source of inoculum, mode of spread and characteristics of lesions on roots of excavated trees; symptom expression in relation to tree age and damage to the root system and years from initial infection to death on trees killed by the fungus. Mortality from A. ostoyae began in both plantations about 5 years after planting, reaching 30% in one and 11% in the other after 35 years. The spatial pattern of mortality was similar to that reported from New Zealand, France and South Africa; however, the temporal pattern differed, beginning later and, instead of declining, continuing at a nearly constant rate to the present. To age 10, nearly all infections were initiated by rhizomorphs; as plantations aged, the proportion of infections occurring at root–root contacts increased. In seven plantations, in moist and wet subzones of the ICH, from 23 to 52% of Douglas‐firs had root lesions, with the higher incidences occurring on moist sites. The occurrence of aboveground symptoms, reduced leader growth and basal resinosis, was related to the percentage of root length colonized by A. ostoyae on trees with more than 30% of root length killed. Average time from infection to death increased from 1 to 2 years at age 6 to 22 years at age 33. The outlook for timber yield from Douglas‐fir plantations in the cedar‐hemlock zone is discussed. Management alternatives for reducing damage from A. ostoyae when regenerating sites are reviewed.