Armillaria jezoensis,a new symbiont ofGaleola septentrionalis (Orchidaceae) in Hokkaido

NineArmillaria isolates obtained from the roots ofGaleola septentrionalis in Hokkaido were identified asA. jezoensis by means of mating tests. Cultures of these isolates were similar in colony morphology, mycelial growth and rhizomorph formation on each of malt extract-dextrose agar (MDA), potato-dextrose agar (PDA), andG. septentrionalis root extractdextrose agar (GDA) media, showing better mycelial growth and rhizomorph formation on GDA medium.     

Biological species and morphological characteristics ofArmillaria mellea complex in Hokkaido:A. sinapina and two new species,A. jezoensis andA. singula

Three intersterility groups ofArmillaria mellea sensu lato were discovered by examining all pairwise combinations of monosporous isolates of basidiomes collected in Hokkaido. One of them, group IV, was identified asA. sinapina by mating it with tester strains. Two new species, groups III and V, were namedA. jezoensis andA. singula, respectively. Their morphological forms and the ecology of their basidiomes are described.     

Changes in Microfungal Communities in Roots of Quercus robur Stumps and their Possible Effect on Colonization by Armillaria

Sixteen fungal communities were sampled by isolating from 0.5–1 cm diameter roots of living trees and stumps of common oak (Quercus robur). The density of fungi was 1.5–2 times greater in roots from stumps than from living trees. The diversity of fungi was similar in living tree roots and stumps. Some of the fungal species with increased densities in stump roots (e.g. Aspergillus kanagawaensis, Chrysosporium pannorum, Cylindrocarpon destructans, C. didymum, Hormiactis candida, Monodictys lepraria, Mycelium radicis atrovirens, Penicillium daleae, P. janczewskii, and Trichocladium opacum) usually stimulated the growth of rhizomorphs of either Armillaria ostoyae or A. gallica in oak‐wood segments in vitro. Eight of 27 isolates that were studied stimulated the rhizomorph growth in both Armillaria spp. It is presumed that the increase in density of `stimulants' may predispose oak stumps to infection by A. ostoyae and A. gallica.     

A note onArmillaria mellea subsp.nipponica subsp. nov. in Japan

Basidiomes of homothallicArmillaria mellea complex were discovered in the forests of Hokkaido. Their monosporous isolates showed partial compatibility with North AmericanA. mellea s. str. haploid testers. The morphological characteristics of basidiomes differed from other temperateA. mellea s. str. in several aspects. Here, JapaneseA. mellea s. str. is reclassified asA. mellea subsp.nipponica and its basidiome morphologies and habitats are described.     

Interaction dynamics between saprobic lignicolous fungi and Armillaria in controlled environments: Exploring the potential for competitive exclusion of Armillaria on peach

Armillaria root rot, caused by Armillaria tabescens and Armillaria mellea, is a major cause of premature tree death in peach orchards in the southeastern United States. The root systems of infected trees can become entirely colonized by Armillaria, serving as an inoculum source for adjacent trees and providing massive inoculum levels in replant situations. If dead or dying trees could be colonized by an effective competitor of Armillaria before their removal, the extent of root colonization by the pathogen could be reduced, thus decreasing the threat to adjacent trees and/or subsequent plantings. Interactions between five species of saprobic lignicolous fungi (Ganoderma lucidum, Hypholoma fasciculare, Phanerochaete velutina, Schizophyllum commune, and Xylaria hypoxylon) and the two Armillaria species were examined in controlled conditions to provide proof of concept for competitive exclusion of Armillaria from peach roots. On agar-coated glass slides, all five potential antagonists induced detrimental reactions in >58% of the Armillaria hyphae observed, with the majority resulting in hyphal swelling or granulation. On poplar wood blocks, all antagonists consistently either overgrew Armillaria colonies or—in the case of S. commune—engaged in deadlock reactions; in all cases, the viability of Armillaria colonies was reduced to <30% of that of unchallenged controls. When inoculated simultaneously onto opposite ends of peach root segments, all antagonists consistently reduced growth and viability of Armillaria on and under the bark, whereby reduction of pathogen growth underneath the bark, Armillaria’s primary ecological niche, was most pronounced for G. lucidum, S. commune, and X. hypoxylon. When root segments were allowed to be colonized entirely by Armillaria before being inoculated with the antagonists, the latter were able to overgrow the pathogen on the root surface but unable to pre-empt it from underneath the bark. In summary,G. lucidum, S. commune, and X. hypoxylon caused strong hyphal and mycelial interference reactions and the most pronounced reductions in growth of Armillaria above and below the bark, indicating that they would be the most promising candidates for field-scale evaluations to restrict colonization of dead or dying peach trees by Armillaria in the orchard.     

Identification ofArmillaria species from hokkaido by analysis of the intergenic spacer (IGS) region of ribosomal DNA using PCR-RFLP

The intergenic spacer (IGS) region, which is located between the 3′ end of 26S ribosomal DNA (rDNA) and the 5′ end of 5S rDNA, of sixArmillaria species from Hokkaido was investigated using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Restriction with onlyAlu I could distinguishA. mellea subsp.nipponica from the other species. WithAlu I andDde I,A. ostoyae andA. gallica could be distinguished from the other species. Digestion withAlu I resulted in two patterns (types A and B) ofA. singula and three patterns (types A, B, and C) ofA. jezoensis. One pattern (type B) of the former species and two patterns (types B and C) of the latter species were each different from those of the other species.Armillaria sinapina gave only oneAlu I digestion pattern, which was identical to that ofA. jezoensis (type A) andA. singula (type A). However, by digestion withDde I,A. singula (type A) could be distinguished fromA. jezoensis (type A) andA. sinapina.     

Studies on ectomycorrhiza

Summary Plants ofPicea abies (L.) Karst were grown in mycorrhizal association withTelephora terrestris (Pers. ex Fr.) andPisolithus tinctorius (Mich. ex Pers.) Coker and Couch on sphagnum peat in petri dishes or Perspex chambers. After 1 yearT. terrestris had formed prominent rhizomorphs which were characterized by light microscopy and investigated for³²P-orthophosphate uptake. The absorbed phosphate was transported to sinks throughout the rhizomorphal system as well as into the plant. The calculated translocation velocity and flux rate in the rhizomorph were in the range of 1–3 cm/h and 0.5–4.0 × 10^-10 mol cm^-2 s^-1, respectively. Label was observed to accumulate in the needles 2–3 days after application. Feeding a non-mycorrhized root with³²P-orthophosphate led to an accumulation of label in needles within 1 h, but no radioactivity appeared in the associatedT. terrestris rhizomorphs. The rhizomorphs ofP. tinctorius revealed a higher structural differentiation than those ofT. terrestris. Translocation of labelled phosphorus through rhizomorphs ofP. tinctorius into spruce needles was also demonstrated.     

Identification of Armillaria species associated with Polyporus umbellatus using ITS sequences of nuclear ribosomal DNA

The sclerotia of Polyporus umbellatus were collected from three locations in Japan and three locations in China. All the collected sclerotia were adhered to by rhizomorphs of the symbionts. When the sclerotium of P. umbellatus was cross sectioned, the internal part of the sclerotium was cream colored, and many black regions surrounding the invading rhizomorphs were observed. The surrounding zone contained string-like, gelatinous masses composed of hyphae, and its outside was brown in color. All isolates were similar in colony morphology and grew well on PDA medium with well-developed rhizomorphs. All the isolates showed typical morphology of Armillaria. The isolated fungi were identified via the ITS region of the nuclear ribosomal DNA sequence. Phylogenetic analysis based on the neighbor-joining method showed that all the isolates clustered with fungi belonging to Armillaria species. Among them, five species (A. sinapina, A. calvescens, A. gallica, A. cepistipes, and A. nabsnona) and the symbiont formed a highly supported clade. We report on the case where Armillaria has a relationship in the sclerotium of Polyporus umbellatus.     

Advances toward DNA-based identification and phylogeny of North American Armillaria species using elongation factor-1 alpha gene

The translation elongation factor-1 alpha (EF-1α) gene was used to examine the phylogenetic relationships among 30 previously characterized isolates representing ten North American Armillaria species: A. solidipes (=A. ostoyae), A. gemina, A. calvescens, A. sinapina, A. mellea, A. gallica, A. nabsnona, North American biological species X, A. cepistipes, and A. tabescens. The phylogenetic relationships revealed clear separation of all ten North American Armillaria species, with the exception of one A. gallica isolate that perhaps represents an unnamed cryptic species. These results indicate that the EF-1α gene could potentially serve as a diagnostic tool for distinguishing among currently recognized North American biological species of Armillaria.     

Effect of mycorrhization on the accumulation of rishitin and solavetivone in potato plantlets challenged with<Emphasis Type="Italic"> Rhizoctonia solani</Emphasis>

The effect of colonization with the vesicular-arbuscular mycorrhizal fungus Glomus etunicatum on the content of rishitin and solavetivone was determined in potato plants cv. Goldrush challenged with Rhizoctonia solani. Mycorrhization stimulated significantly the accumulation of both phytoalexins in roots of plantlets challenged with R. solani but did not influence phytoalexin levels in non-challenged plantlet roots. No accumulation of solavetivone or rishitin was detected in shoots. In Petri dish bioassays, rishitin and solavetivone inhibited mycelial growth of R. solani.     

Molecular phylogeny of Armillaria from the Patagonian Andes

A number of species in the plant pathogen genus Armillaria are known from South America where they cause root rot disease on a wide variety of hosts. Knowledge pertaining to phylogenetic relationships of these species with those of other Armillaria species is almost non-existent. In addition, very few cultures representing these species are available, making DNA-based phylogenetic analyses impossible. The aim of this study was to characterise a collection of Armillaria isolates from the Patagonian Andes using DNA sequences and to determine their phylogenetic relationships with other Armillaria species. DNA sequences were obtained from the internal transcribed regions (ITS1, 5.8S and ITS4) and ribosomal large subunit (LSU) gene and used in phylogenetic analyses. Phylogenetic trees generated from the sequences separated the Armillaria isolates into four lineages. Lineages I and II represented A. novae-zelandiae and A. luteobubalina, respectively. Isolates belonging to A. novae-zelandiae from Malaysia, New Zealand, Australia and South America showed considerable intra-clade sub-structure. Lineages III and IV are probably distinct species and are most closely related to A. hinnulea and an unnamed species isolated from New Zealand and Kenya. This is the first comprehensive study of the phylogenetic relationships of Armillaria species from Patagonia and it provides a foundation for future research in this region.     

Stand structure and growth patterns of understorey trees in a coniferous forest,Taisetsuzan National Park,northern Japan

Stand structure was studied with special reference to growth and mortality patterns of sapling and understorey trees in a coniferousPicea jezoensis andAbies sachalinensis forest in Taisetsuzan National Park, Hokkaido, northern Japan.Picea jezoensis was dominant in the basal area, whileA. sachalinensis was abundant in large numbers in the canopy. Estimated mortalities increased significantly with diameter at breast height (DBH) for bothP. jezoensis andA. sachalinensis in the canopy, but the tendency was different between the two species.Picea jezoensis had a lower mortality rate thanA. sachalinensis, especially at small DBH classes. The spatial distribution of understorey individuals ofA. sachalinensis did not show any significant correlation with the spatial distribution of canopy gaps, but that ofP. jezoensis showed a significant correlation.Abies sachalinensis can grow higher thanP. jezoensis under suppressed conditions; whileP. jezoensis requires canopy gaps for steady height growth. This growth pattern leads to a different waiting height in the understorey (≥2 m in height and 10 cm in diameter at breast height).Abies sachalinensis waited for an improvement in light conditions at higher strata (max. 7 m), whileP. jezoensis waited at lower strata (max. 3 m). The estimated mortality of understoreyA. sachalinensis increased with size, while that of understoreyP. jezoensis decreased. Therefore,P. jezoensis gives priority to survival whileA. sachalinensis gives priority to understorey growth. The difference in the ‘waiting pattern’ between the two species in the understorey was considered a significant feature for the canopy recruitment process ofP. jezoensis andA. sachalinensis.     

Comparative bioluminescence dynamics among multiple Armillaria gallica, A. mellea, and A. tabescens genets

Bioluminescence is well known among white-spored species of Basidiomycota including several species of the white-rot wood decay genus Armillaria. Previous work demonstrated consistent differences among A. gallica, A. mellea, and A. tabescens in luminescence magnitude and in luminescence expression relative to environmental stimuli. In the present studies, temporal fluctuations in mycelial luminescence were quantitatively characterized using genets matched for geographical location. All genets derived from rhizomorphs or basdiomata were constitutively luminescent while six of 13 genets originating from mycelial fans were inconsistently luminescent. Using time series of 1000 consecutive measurements over 800 ms intervals, fluctuation patterns had significantly quantifiable structure and were not simply ‘white noise’. Fluctuation patterns were qualitatively similar with alternating periods of rapid fluctuation and relative stability, regardless of luminescence magnitude. Anomalous spikes or shifts in luminescence were recorded for several genets suggesting further work to identify the transient stimuli which elicited these altered luminescence patterns.     

Armillaria luteobubalina mycelium develops air pores that conduct oxygen to rhizomorph clusters

Armillaria luteobubalina produces air pores in culture. They consist of two parts: a basal region of tissue elevated to form a mound covered with a rind continuous with that of the colony, but perforated; and an apical region of long parallel hyphae, cemented together by scattered patches of extracellular material. This forms a hydrophobic structure that is elevated above the general level of the mycelial crust and does not easily become waterlogged. Air pores develop near the inoculum plug shortly after inoculation, arising directly from the mycelium, and rhizomorphs are initiated from them. The air pore contains a complex system of gas space connecting the atmosphere with the central canal of each rhizomorph. The tissue beneath the melanised colony crust also contains gas space, especially near air pores. This is also connected with the gas space of each rhizomorph and of each air pore. Measurements with oxygen electrodes show that air pores and their associated rhizomorphs conduct oxygen. The average oxygen conductance of a group of air pores with associated rhizomorphs, within agar blocks, but with rhizomorph apices cut off, was about 700 × 10⁻¹² m³ s⁻¹, equivalent to about 200 × 10⁻¹² m³ s⁻¹ for each air-pore. We conclude that the air pores conduct oxygen into the gas space below the pigmented mycelium of the colony, where the rhizomorphs - which also conduct oxygen - originate. A. luteobubalina thus has a complex aerating system which allows efficient diffusion of oxygen into rhizomorphs, and this is likely to facilitate extension of inoculum into low-oxygen environments.     

Characterization of Armillaria spp. from peach orchards in the southeastern United States using fatty acid methyl ester profiling

Limited information is available regarding the composition of cellular fatty acids in Armillaria and the extent to which fatty acid profiles can be used to characterize species in this genus. Fatty acid methyl ester (FAME) profiles generated from cultures of A. tabescens, A. mellea, and A. gallica consisted of 16–18 fatty acids ranging from 12–24 carbons in length, although some of these were present only in trace amounts. Across the three species, 9-cis,12-cis-octadecadienoic acid (9,12-C18:2), hexadecanoic acid (16:0), heneicosanoic acid (21:0), 9-cis-octadecenoic acid (9-C18:1), and 2-hydroxy-docosanoic acid (OH-22:0) were the most abundant fatty acids. FAME profiles from different thallus morphologies (mycelium, sclerotial crust, or rhizomorphs) displayed by cultures of A. gallica showed that thallus type had no significant effect on cellular fatty acid composition (P > 0.05), suggesting that FAME profiling is sufficiently robust for species differentiation despite potential differences in thallus morphology within and among species. The three Armillaria species included in this study could be distinguished from other lignicolous basidiomycete species commonly occurring on peach (Schizophyllum commune, Ganoderma lucidum, Stereum hirsutum, and Trametes versicolor) on the basis of FAME profiles using stepwise discriminant analysis (average squared canonical correlation = 0.953), whereby 9-C18:1, 9,12-C18:2, and 10-cis-hexadecenoic acid (10-C16:1) were the three strongest contributors. In a separate stepwise discriminant analysis, A. tabescens, A. mellea, and A. gallica were separated from one another based on their fatty acid profiles (average squared canonical correlation = 0.924), with 11-cis-octadecenoic acid (11-C18:1), 9-C18:1, and 2-hydroxy-hexadecanoic acid (OH-16:0) being most important for species separation. When fatty acids were extracted directly from mycelium dissected from naturally infected host tissue, the FAME-based discriminant functions developed in the preceding experiments classified all samples (n = 16) as A. tabescens; when applied to cultures derived from the same naturally infected samples, all unknowns were similarly classified as A. tabescens. Thus, FAME species classification of Armillaria unknowns directly from infected tissues may be feasible. Species designation of unknown Armillaria cultures by FAME analysis was identical to that indicated by IGS-RFLP classification with AluI.     

Lifespans of fungal rhizomorphs under nitrogen fertilization in a pinyon-juniper woodland

The lifespan of individual microbes in the soil influences nutrient cycling rates as well as population dynamics, but their responses to global change factors such as anthropogenic nitrogen deposition have been challenging to quantify in situ. We used minirhizotron images to track the abundance and turnover rate of individual fungal rhizomorphs under nitrogen fertilization in a pinyon-juniper woodland in New Mexico. We hypothesized that increases in nitrogen availability would alter rhizomorph lifespan and abundance. Sequential images were collected over eight sampling dates from November 1997 to August 1999, and a total of 278 rhizomorphs were examined. We found that neither standing stocks nor lifespans of rhizomorphs differed significantly between treatments. Lifespans of rhizomorphs lasted eleven months on average, indicating that nutrient immobilization in these structures could last for longer than a growing season in these sites.     

The Effect of Felling on the Occurrence of Microfungi Stimulatory to Armillaria Rhizomorph Formation in Thin Roots of Quercus robur

Eight fungal assemblages were isolated from thin branch roots (0.5–1 mm diameter) of living oak trees (Quercus robur) and their stumps 2 years after felling. The roots were serially washed or surface‐sterilized with ethanol and hypochlorite. The thin roots were inhabited by fungi that are stimulants of Armillaria rhizomorphs as found previously in thick roots (0.5–1 cm diameter). However, unlike thick roots, the densities of the fungi in serially washed roots were significantly less in stump roots than in living roots. Among the most common ‘stimulants’, the frequencies of Cylindrocarpon didymum, Mortierella gracilis, Mor. microspora var. macrocystis, Mycelium radicis atrovirens, Nectria grammicospora, Pseudogymnoascus roseus and Sporothrix schenckii were usually significantly greater in stump roots than in living roots, while the frequencies of Chrysosporium merdarium, Cy. destructans, Mor. hygrophila, Penicillium adametzii, Pe. citrinum, Pe. daleae, Pe. janczewskii and Pe. spinulosum were usually significantly less in stump roots. The frequency of Trichoderma viride was less in the stump roots. Serial washing of roots was more effective than chemical surface‐sterilization in the detection of Armillaria rhizomorph ‘stimulants’. The significance of these findings in the ecology and risk assessment of Armillaria in plantations is discussed.     

Diversity and ecology of <Emphasis Type="Italic">Armillaria</Emphasis> species in virgin forests in the Ukrainian Carpathians

In this study, we investigated the diversity and ecology of Armillaria species in virgin pure beech and mixed conifer forests (15,000 ha) of the Carpathian Biosphere Reserve in Ukraine. Armillaria rhizomorphs were systematically sampled, both from the soil and from the root collar of trees (epiphytic), on 79 plots (25 × 20 m) of a 1.5 × 1.5 km grid. In both forest massifs, rhizomorphs were present in the majority of the soil samples, with an estimated dry weight of 512 kg/ha in the pure beech forests and 223 kg/ha in the mixed conifer forests. Similarly, in both forest massifs, most of the trees inspected had rhizomorphs at the root collar. Species identification based on DNA analyses showed that all five annulated European Armillaria species occur in these virgin forests, as previously observed in managed forests in central Europe. However, differences in the frequencies of the single species were observed. The predominance of the preferentially saprotrophic A. cepistipes and A. gallica (84 and 15% of the specimens, respectively) and the absence of significant pathogenic activity suggest that in these virgin forests Armillaria species are most likely to behave as saprotrophs. Forest management may increase the frequency of the pathogenic species A. ostoyae, which is rare in virgin forests.     

Rhizospheric streptomycetes as potential biocontrol agents of <Emphasis Type="Italic">Fusarium</Emphasis> and <Emphasis Type="Italic">Armillaria</Emphasis> pine rot and as PGPR for <Emphasis Type="Italic">Pinus taeda</Emphasis>

Pinus taeda is one of the main timber trees in Brazil, occupying 1.8 million ha with an annual productivity of 25–30 m³ ha⁻¹. Another important species is Araucaria angustifolia, belonging to the fragile Rainforest biome, which for decades has been a major source of timber in Brazil. Some diseases that affect the roots and/or the stem of these trees and cause “damping-off” of the seedlings, with economic and environmental losses for the forest sector, are caused by the plant pathogenic fungi Fusarium sp. or Armillaria sp. This research project intended to isolate actinobacteria from the Araucaria rhizosphere, which present an antagonistic effect against these fungi. After the selection of the best pathogen inhibitors, morphologic characteristics, enzyme production, and their effect on the growth of Pinus taeda were studied. The actinobacteria were tested for their antagonistic capacity against Fusarium sp. in Petri plates with PDA as substrate. The inhibition zone was measured after 3, 5, 7, and 10 days. Of all the isolates tested, only two of them maintained inhibition zones up to 4 mm for 10 days. The inhibition of Armillaria sp. was tested in liquid medium and also in Petri dishes through the evaluation of the number of the fungal rhizomorphs in dual culture with the actinobacteria. It was found that all five isolates were able to inhibit the rhizomorph production, with the best performance of the isolate A43, which was capable of inhibiting both fungi, Fusarium and Armillaria. In a greenhouse experiment, the effect of five isolates on the growth of Pinus taeda seedlings was tested. Plant height, stem diameter, root and shoot dry matter were determined. The Streptomyces isolate A43 doubled plant growth. These results may lead to the development of new technologies in the identification of still unknown bacterial metabolites and new management techniques to control forest plant diseases.     

Morphological and molecular characterization of the Armillaria cepistipes – A. gallica complex in the Czech Republic and Slovakia

Armillaria cepistipes and A. gallica (Basidiomycota, Physalacriaceae) are morphologically similar species, and they are often nearly indistinguishable using DNA-based methods targeting the ITS region of ribosomal DNA. The aim of this study was to examine morphological and ecological features of A. cepistipes and A. gallica, and to test other DNA-based methods to distinguish the two species. Our results revealed discriminative macro- and micromorphological features between these two species, especially the presence of a distinct central pileus ocella, the shape of the annulus, the character of the velar stipe remnants and the length of the terminal cells of the pileus scales. Ecologically, A. gallica generally prefers warmer areas in lowlands (oak and alluvial forests), while A. cepistipes is more common in hilly and lower montane beech forests in Central Europe. Nevertheless, despite differences in ecological preferences, certain locations between 300 and 500 m a.s.l. are known to sympatrically support both species. The sequences of the translation elongation factor 1-alpha showed high interspecific variability, and this gene is a more appropriate candidate for distinguishing A. gallica from A. cepistipes.