Effect of nitrogen and carbon sources on lignin and cellulose degradation by Armillaria ostoyae

Lignin has been hypothesized to be the primary mechanism of resistance to fungal pathogens in plant tissue. Degradation of lignin and cellulose by Armillaria ostoyae cultured for six weeks in Melin-Norkrans medium containing various nitrogen and carbon sources was measured radiometrically. No consistent pattern of lignin or cellulose degradation was found, regardless of A. ostoyae isolate, nitrogen source and concentration, or carbon concentration. More lignin was degraded as the concentration of glucose and fructose increased but not when the concentration of sucrose increased.     

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).     

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.     

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.     

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.     

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.     

Stimulation of Armillaria mellea growth by plant hormones in relation to the concentration and type of carbohydrate

Thalli of Armillaria mellea grew vigorously and produced abundant rhizomorph after 21 days of incubation on a glucose-L-asparagine medium supplemented with auxin, but grew poorly and failed to produce rhizomorphs on a non-supplemented medium or on one supplemented with either gibberellic acid or kinetin. Replacement of glucose in auxin-supplemented medium with arabinose, fructose, galactose, maltose, sucrose or starch resulted in poor thallus growth and a lack of rhizomorph development. The superiority of glucose as a carbon source for thallus growth appears to be related to its superiority as a substrate for metabolism by A. mellea.     

Phylogenetic analysis of Japanese Armillaria based on the intergenic spacer (IGS) sequences of their ribosomal DNA

To determine the phylogenetic positions of two new species, Armillaria jezoensis and Armillaria singula, and one new subspecies, Armillaria mellea suhsp. nipponica, the nucleotide sequences of the intergenic spacers (IGS) of their ribosomal DNA were investigated, and compared with those of tour other Armillaria species from Japan, and those of nine Armillaria species from Europe and North America. We conclude that Armillaria jezoensis, and Armillaria singula belong to the Armillaria gallica cluster as Armillaria cepistipes, Armillaria gallica and Armillaria sinapina from Japan. Two isolates of Armillaria ostoyae from Japan were placed within the Armillaria ostoyae cluster. Armillaria mellea subsp. nipponica had an IGS sequence as long as the IGS of Armillaria mellea from Europe and North America. However, the IGS sequences of Armillaria mellea subsp. nipponica, whose basidium base lacks a clamp connection could not be satisfactorily aligned with the IGS sequences of other species possessing this morphological feature.     

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.     

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.     

Development and verification of a diagnostic assay based on EF‐1 α for the identification of Armillaria species in Northern Europe

The overall aim of this study was to develop a new, reliable and rapid diagnostic assay for differentiating six European Armillaria species based on variation in their elongation factor‐1 alpha (EF‐1 α) gene sequences and to verify a set of species‐specific primers on 61 Armillaria isolates from Europe. Partial sequences of the EF‐1 α gene obtained in Armillaria borealis, Armillaria cepistipes, Armillaria gallica, Armillaria mellea, Armillaria ostoyae and Armillaria tabescens revealed sufficient interspecific variation to distinguish among species using nested primers. These primers gave unambiguous bands when tested on representative isolates of five of these species. However, the EF‐1 α sequences of European A. borealis isolates clustered into two distinct clades, termed here AbX and AbY. Specific primers were subsequently designed and tested successfully on both AbX‐type and AbY‐type A. borealis isolates. The taxonomy of A. borealis needs to be elucidated to determine whether a new, as yet unnamed Armillaria taxon exists in Europe. Three A. borealis isolates were also found to have heterozygous sites in their EF‐1 α sequences, which suggests that the gene could exist in more than one copy or that these isolates contain hybrid sequences. A pyrosequencing method was also developed, targeting a small region of EF‐1 α intron 4, which was able to differentiate European Armillaria isolates to the species level and additionally could distinguish AbX‐type and AbY‐type A. borealis isolates.     

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.     

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.     

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.     

Comparaison de deux méthodes d'identification des clones chèz le Basidiomycete parasite Armillaria obscura (syn: A. ostoyae)

Two methods were compared intending to distinguish between the clones of the root-rot fungus Armillaria obscura (syn. Armillaria ostoyae) in nature: one based on the pairing of the diploid isolates in pure culture, the other using the incompatibility alleles as genetic markers. As A. obscura fruits easily in vitro, both methods could be used for any type of diploid isolate whatever its origin. The two methods were quite superposable and defined the same clones. Artificial diploids issuing from the same fruitbody and isogenic for the incompatibility alleles appeared to belong to the same clone in pairing tests.     

Relationship of site and stand characteristics to Armillaria root disease incidence on ponderosa pine in the Black Hills,South Dakota

Ponderosa pine (Pinus ponderosa) in the Black Hills National Forest, SD, USA, was surveyed for Armillaria root disease (ARD). The root pathogen Armillaria ostoyae occurred on ponderosa pine seedlings, saplings, pole‐size trees and large‐diameter trees. The mean incidence of aboveground disease symptoms by stem count was low (0.2%), but in certain areas, the incidence was higher, affecting the regeneration success and tree longevity. Symptomatic ponderosa pine were in areas characterized by having higher elevation, greater annual precipitation, more seedlings, bigger large‐diameter trees and greater odds of past harvesting activity than in areas without root disease. Stump density was positively spatially correlated with root disease incidence. No particular soil type was related to disease occurrence; though, in areas with symptomatic trees, soil available water holding capacity (AWC) was greater and soil permeability was less where root disease was present. Spatial analysis confirmed the relationships found in linear correlations, with soil AWC and stump density positively and soil permeability negatively correlated with per cent infected stems ha^?1 and basal area infected.     

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.     

Screening and verification of the factors influencing somatic embryo maturation of <Emphasis Type="Italic">Larix olgensis</Emphasis>

With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo. Three dominant factors emerged, and we developed a response surface model based on the Box–Behnken design. We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204?±?4 g^?1 on basal medium, containing 18.28 mg L^?1 abscisic acid, 5.46 mg L^?1 silver nitrate and 82.67 g L^?1 sucrose. In the verification experiments, the addition of 20 mg L^?1 abscisic acid, 5 mg L^?1 silver nitrate and 80 g L^?1 sucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis.     

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.     

外源糖溶液和高浓度CO2处理对白桦叶片糖和蛋白质含量的影响

3年生白桦同时接受3种外源糖溶液(蔗糖、果糖、葡萄糖)和3种高浓度CO2(700、1400、2100μL·μL-1CO2)处理.处理1个月后,测定了叶片的总糖、蔗糖、果糖和蛋白质含量.结果表明:在700μL·L-1和1400μL·L-1 CO2下,外源糖溶液增加了叶片的可溶性糖和蛋白质含量,其中外源蔗糖的效果最好:外源糖溶液与2100μL·L-1CO2结合,会抑制叶片积累总糖和蛋白质:在700μL·L-1和1400μL·L-1CO2下,喷施葡萄糖、果糖的叶片在蛋白质含量上没有明显差别:同700、1400μL·L-1CO2相比,除喷施果糖植株外,2100μL·L-1 CO2明显增加了叶片的总糖、蔗糖、果糖和蛋白质含量:在喷施同种外源糖溶液的情况下,叶片的糖含量与CO2浓度呈正相关性.图6参7.