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Several methods for fruiting Heterobasidion annosum are presented. The methods are simple, reproducible, and can be applied to heterokaryotic mycelia isolated from nature as well as the mycelia resulting from crosses of homokaryotic strains. Methods for mutagenesis and recovery of auxotrophic and morphological mutants are discussed. Instances of fruiting in common-^ heterokaryons and in homokaryons are analyzed. The importance of meeting genetic criteria for successful fruiting is discussed. Heterobasidion annosum (Fr.) Bref. [-Fomes annosus (Fr.) Karst.] causes root decay in coniferous trees. A voluminous literature describes the fungus (see Koenigs, 1960, and Hodges, 1969, for reviews). Few reports consider the genetics of H. annosum. This is understandable because of the scarcity of requisite infor? mation on the mating system and reliable methods to promote sexual fruiting. Recent reports (Korhonen, 1978; Chase and Ullrich, 1983) satisfy essential needs for rudimentary information on the mating system. Heterobasidion an? nosum is heterothallic, multiallelic and unifactorial except for homothallic isolates from Australia (Chase, unpubl. data). Reports on the fruiting of H. annosum in laboratory culture are infrequent. Reynolds (1973), Miller (1960), and Boyce (1962) developed nonaxenic methods of fruiting H. annosum. These involve the use of logs which contain H. annosum incubated under moist but nonsterile conditions. These methods are not wellsuited to genetic analysis. There have been few reports of fruiting of cultures of H. annosum under axenic conditions. Liese (1931) obtained fruiting of H. an? nosum on malt extract agar following exposure of cultures to temperatures less than -26 C. Sychev and Negrutskii (1978) obtained fruiting on White's medium, wort agar, and on various types of wood. Cartwright and Findlay (1958) observed occasional fruiting on sterilized wood blocks. Siepmann (1970) induced H. an? nosum to fruit on sterile wood under controlled environmental conditions. Most recently Holt et al (1983) developed a method for fruiting cultures isolated from infected roots and wood, as well as from mycelia resulting from crosses of ho? mokaryotic strains in the laboratory. This method involves a complex medium and long (up to six months) incubation periods. Except for the report of Holt et al (1983) the earlier papers have not included data to permit assessment of the reproducibility of their particular methods. None has utilized a large, diverse group of H. annosum isolates and fruiting has usually occurred only on very complex media. During the course of our genetic studies (Chase and Ullrich, 1983) we observed that many independent heterokaryons of H. annosum fruited when inoculated to standard 1.25% malt extract agar in Petri dishes. We report improvements in this procedure that permit routine genetic analysis in H. annosum.

Armillaria mellea ss. lato and Heterobasidion annosum were identified in infected Norway spruce using an indirect immunohistochemical test. Unspecific binding sites were masked by incubating the specimen with preimmune serum followed by protein A. Subsequent labeling with monospecific rabbit antibodies and fluorochrome-conjugated protein A yielded a strong and specific fluorescent signal.

SUMMARY Inoculation of pine stumps with oidia of Peniophora gigantea directly after felling gives good protection against Fomes annosus. With the largest inoculum of F. annosus spores likely to occur naturally, a dosage of 1 × 104oidia suffices for stumps having a wood-diameter of 16 cm. P. gigantea enters the lateral roots of inoculated stumps and tends to check advance of F. annosus in any tissues infected at the time of felling; it also replaces this fungus to some extent, Lignin is rapidly decomposed and sporophores are generally produced within a year. Suspensions may conveniently be applied by a compressed air sprayer. Owing to the selectivity of freshly cut stumps, contamination of the suspension is not a serious hazard. If widely used, the method would necessitate large-scale production of suitable inoculum sources. For most of the experimental work undertaken the inoculum was derived from wood-block cultures of P. gigantea stored in polythene bags, but for routine use a more compact source would be preferable. A method is described for preparing tablets which, when dissolved in IOO ml. water, yield suspensions containing up to 1 × 106viable oidia per ml.: if kept at 10° C. they should each provide inoculum for more than a hundred stumps over a storage period of 10–20 weeks. The relative merits of inoculation and chemical treatment for control of F. annosus in pine stumps are briefly discussed and other possible applications of stump inoculation are outlined.

The following genera are redefined:Albatrellus S. F. Gray,Heterobasidion Bref.,Haploporus Bond. et Sing. ex Sing.,Fomitopsis P. Karst. andRigidoporus Murrill two new subgenera are described:Polyporus subgen.Dendropolyporus Pouz. (type:Polyporus umbellatus) andRigidoporus subgen.Neooxyporus Pouz. (type:Polyporus latemarginatus); the genusOxyporus (Bourd. etGalz.)Donk is classified as a subgenus of the genusRigidoporus,Murrill and the generaBjerkandera P. Karst. andLeptoporus quel. are classified as subgenera of the genusTyromyces P. Karst. The new subfamilyAlbatrelloideae Pouz. (genera:Albatrellus andGrifola) is described and 14 new specific combinations are made. The new genusIrpicodon Pouz. (type:Irpex pendulus) is proposed.

The root rot pathogens in Norway spruce (Picea abies) Heterobasidion ssp. cause substantial loss in carbon sequestered in forest and economic revenue for forest owners. To facilitate strategic breeding planning for increased resistance against this pathogen in particular, the blue stain fungus Endoconidiophora polonica, growth and wood quality traits (wood density and spiral grain), we estimated additive genetic parameters, correlations and the potential response from selection. Parameters were estimated from a progeny trial series established at two sites (25 years from planting) and their parents in a seed orchard (43 years from grafting). A standard half-sib analysis based on progenies and a parent–offspring regression was used for estimation of heritabilities. Resistance against the pathogens was measured as lesion length under bark after inoculations in phloem. Heritability values varied with site and estimation procedure from 0.06 to 0.33, whereas the phenotypic variance (as CV P ) is high and fairly stable around 40–50 %. Heritability values for wood density and spiral grain in the same material varied from 0.32 to 0.63. The highest heritability values were generally obtained from parent–offspring regression. There is no evidence of resistance traits being genetically correlated with growth or wood quality traits. Wood density is negatively correlated with stem diameter. Implications for breeding are discussed.

Management practices over the past 100 years, especially fire exclusion and suppression, in Yosemite Valley, Yosemite National Park, CA resulted in forest stands with basal areas in excess of 110 m2 ha-1. Accelerated attack by several species of bark beetles (Scolytidae) followed a severe drought in 1976–77. Pinus ponderosa, already under attack by root rot (Heterobasidion annosus) was affected severely, and mortality exceeded 70%. Calocedrus decurrens did not suffer comparable mortality and has replaced P. ponderosa as the dominant species. Size class distributions as well as comparisons of relative density and relative basal area from 1961, 1976 and 1983–85 also demonstrate a major change in forest composition. This change is most pronounced on alluvial landforms.

This study provides new information on the infection biology and pathogenicity of an important root-rot fungus, Heterobasidion annosum sensu stricto (Fr.) Bref., through a detailed examination of the vegetative spread of clonal individuals and their capacity to produce fruiting bodies on young pine seedlings. The seedlings were planted in a clear-cutting area (c. 1.2 ha in size) after a pine generation that showed slight external symptoms of Heterobasidion root rot. The first dead seedlings were found five years after planting and during a nine-year monitoring period; nearly 600 seedlings were killed by H. annosum s.s. in 48 individual disease centers. Based on pairing tests of 482 isolates, 117 different H. annosum s.s. genotypes were identified. On average, 2.9 genotypes occurred in a single disease center. The extensive secondary spread of genotypes within root systems (up to 48 pine seedlings infected by the same genotype) resulted in annually expanding disease centers. In addition, more than half of the seedlings killed by H. annosum s.s. produced perennial fruiting bodies thus providing air-borne inoculum. The risk of spore infection should be taken into account in any type of cutting operation in young pine stands. Moreover, new control measures directed towards the secondary spread of H. annosum s.s. in pine regeneration are urgently needed in order to maintain the productivity of the pine forest on infested sites.

Research Highlights: Dedicated experiments to investigate how disturbances will affect Heterobasidion sp.—Norway spruce pathosystems are important, in order to develop different strategies to limit the spread of Heterobasidion annosum s.l. under the predicted climate change. Here, we report on a greenhouse experiment to evaluate the effects of water availability on the infection severity of Heterobasidion parviporum or Heterobasidion annosum, respectively, on Picea abies saplings. Background and Objectives: Changes in climatic conditions and intense logging will continue to promote H. annosum s.l. in conifer forests, increasing annual economic losses. Thus, our aim was to test if disease severity in Norway spruce was greater after infection with H. parviporum or H. annosum in low water availability conditions, compared to seedlings with high water availability. Materials and Methods: We performed inoculation studies of three-year-old saplings in a greenhouse. Saplings were treated as high (+) or low (−) water groups: High water group received double the water amount than the low water group. The necrosis observed after pathogen inoculation was measured and analyzed. Results: The seedling growth was negatively influenced in the lower water group. In addition, the water availability enhanced the necrosis length of H. parviporum in phloem and sapwood (vertical length) in the low water group. H. annosum benefited only in horizontal length in the phloem. Conclusions: Disturbances related to water availability, especially low water conditions, can have negative effects on the tree host and benefit the infection ability of the pathogens in the host.

Summary The xylem surface of seedlings, stem material and roots of Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) were inoculated with strains of Heterobasidion annosum s. str. and H. parviporum s. str. The depth of necrosis in wounded spruce increased at a linear rate for at least seven weeks of incubation, but the rate of necrotic spread was significantly faster in infected wounds. In wounded pine the necrosis was maintained at a more superficial level for several weeks. Both spruce and pine sapwood were initially infected by hyphae of both species. In spruce, the hyphae advanced at a constant rate behind the necrotic front. On the contrary, after 1–2 weeks living H. parviporum hyphae were rare in pine rays. Heterobasidion annosum hyphae survived in pine rays, phloem and tracheids, despite a heavy accumulation of phenolics and resins and were able to penetrate into the sapwood at a linear rate although slower than infections in spruce. Histochemistry and quantitative estimates demonstrated that peroxidase activity was initially higher in spruce sapwood than in pine. Within three days of incubation, the activity in spruce sapwood disappeared concurrently with deepening necrosis. However, in pine, in both control and infected samples, there was a significant increase in peroxidase activity in the area surrounding the superficial necrosis, up to the wound surface and in the cambium and phloem around the wound. After wounding and infection, the content of soluble protein increased significantly in wood of older trees but not in seedlings. Infection resulted in an increased formation of lipophilic extractives in both spruce and pine but to a significantly greater degree in the latter, whereas the amount of hydrophilic compounds decreased in both. High-performance liquid chromatography (HPLC) analyses of lipophilic extracts showed that inoculation of pine with the two species of Heterobasidion increased the amounts of pinosylvin, its monomethylether and several other phenolics as also resinous compounds. The results obtained may be relevant in explaining the known higher resistance of Scots pine to H. parviporum. Resume Des semis, tiges et racines d'epicea commun (Picea abies) et de pin sylvestre (Pinus sylvestris) ont ete inocules au niveau du cambium avec des souches d’Heterobasidion annosum (Fr.) Bref. et Heterobasidion parviporum Niemela & Korhonen. La profondeur de necrose a partir des blessures a presente une augmentation lineaire pendant au moins sept semaines d'incubation chez les epiceas, mais l'extension de la necrose a ete significativement plus rapide pour les blessures infectees. Dans le cas des blessures sur pins, la necrose s'est limitee a un niveau superficiel pendant plusieurs semaines. Aussi bien chez les pins que les epiceas, l'aubier a commenceaetre infecte par les hyphes des deux especes d’Heterobasidion. Chez les epiceas, les hyphes ont progressea un taux constant derriere le front de necrose. Au contraire, les hyphes vivants de H. parviporumetaient rares dans les rayons apres une a deux semaines chez les pins. Les hyphes de H. annosum ont survecu dans les rayons, phloeme et tracheides de pins malgre une intense accumulation de resines et phenols et ont pu penetrer dans l'aubier, a un taux lineaire plus faible que dans le cas des infections sur epicea. Une etude histochimique et des estimations quantitatives ont montre que l'activite peroxydase etait initialement plus forte dans l'aubier d'epicea que sur pin. Apres 3 jours d'incubation, l'activite dans l'aubier d'epicea a disparu, en meme temps que la necrose devenait plus profonde. Au contraire chez les pins, aussi bien temoins qu'infectes, l'activite peroxydase a significativement augmente dans la zone entourant la necrose superficielle jusqu’a la blessure de surface, et dans le cambium et le phloeme autour de la blessure. Le contenu en proteins solubles a augmente significativement apres blessure et infection dans le bois des arbres mais pas chez les semis. L'infection s'est traduite par une baisse des composes hydrophiles chez les deux especes et par la formation accrue d'extraits lipophiles, a un niveau toutefois plus important chez les pins. Les analyses par HPLC des extraits lipophiles ont montre que l'inoculation des pins avec les deux especes d’Heterobasidion s'est traduite par une augmentation des teneurs en pinosylvine, de son monomethylether et de plusieurs autres composes phenoliques et resiniques. Les resultats obtenus pourraient expliquer la plus grande resistance, prealablement connue, du pin sylvestre aHeterobasidion parviporum. Zusammenfassung Die Xylemoberflache von Samlingen, Stammabschnitten und Wurzeln der Fichte (Picea abies) und Kiefer (Pinus sylvestris) wurden mit Heterobasidion annosum s.str. und H. parviporum inokuliert. Bei Fichten, die nur verletzt waren, nahm die Tiefe der Nekrose wahrend mindestens 7 Wochen linear zu; in infizierten Wunden breitete sich die Nekrose dagegen signifikant schneller aus. In verletzten Kiefern blieb die Nekrose fur mehrere Wochen auf einen oberflachennahen Bereich beschrankt. Beide Baumarten wurden kurz nach der Inokulation von Hyphen beider Heterobasidion-Arten infiziert. Bei der Fichte breiteten sich die Hyphen mit einer konstanten Wachstumsrate hinter der nekrotischen Front aus. Bei der Kiefer wurden lebende Hyphen von H. parviporum dagegen nach 1–2 Wochen nur selten in den Holzstrahlen gefunden. Die H. annosum-Hyphen uberlebten in den Holzstrahlen, im Phloem und in den Tracheiden der Kiefer trotz einer starken Akkumulation von Phenolen und Harzen, und sie konnten sich im Kiefern-Splintholz zwar langsamer als in der Fichte, aber doch mit einer linearen Wachstumstrate ausbreiten. Histochemische Reaktionen und quantitative Analysen zeigten, dass die Peroxidaseaktivitat im Splintholz der Fichte anfangs hoher warals in dem der Kiefer. Bei der Fichte verschwand diese Aktivitat innerhalb von 3 Tagen und gleichzeitig entwickelte sich eine Nekrose. Bei der Kiefer wurde dagegen sowohl in der Kontrolle als auch in den infizierten Proben ein deutlicher Anstieg der Peroxidaseaktivitat in der Umgebung der oberflachlichen Nekrose bis zur Wundoberflache sowie im Kambium und im Phloem in Wundnahe beobachtet. Nach Verletzung und Inokulation stieg der Proteingehalt im Holz alterer Baume signifikant an, bei Samlingen war dies jedoch nicht der Fall. Die Infektion fuhrte auch zu einer verstarkten Bildung von lipophilen Extraktstoffen (bei Kiefer signifikant mehr als bei Fichte), wahrend die hydrophilen Verbindungen bei beiden Baumarten abnahmen. HPLC-Analysen der lipophilen Extrakte zeigten, dass bei der Fichte durch beide Heterobasidion-Arten die Bildung von Pinosylvin, seinem Monomethylather, verschiedenen Phenolverbindungen und von harzartigen Verbindungen deutlich erhoht wurde. Diese Befunde konnen zur Erklarung der hoheren Resistenz der Kiefer gegen H. parviporum beitragen.

The major threats to the sustainable supply of forest tree products are adverse climate, pests and diseases. Climate change, exemplified by increased drought, poses a unique threat to global forest health. This is attributed to the unpredictable behavior of forest pathosystems, which can favor fungal pathogens over the host under persistent drought stress conditions in the future. Currently, the effects of drought on tree resistance against pathogens are hypothetical, thus research is needed to identify these correlations. Norway spruce (Picea abies (L.) H. Karst.) is one of the most economically important tree species in Europe and is considered highly vulnerable to changes in climate. Dedicated experiments to investigate how disturbances will affect the Norway spruce—Heterobasidion sp. pathosystem are important, in order to develop different strategies to limit the spread of H. annosum s.l. under the predicted climate change. Here, we report a transcriptional study to compare Norway spruce gene expressions to evaluate the effects of water availability and the infection of Heterobasidion parviporum. We performed inoculation studies of three-year-old saplings in a greenhouse (purchased from a nursery). Norway spruce saplings were treated in either high (+) or low (−) water groups: high water group received double the water amount than the low water group. RNA was extracted and sequenced. Similarly, we quantified gene expression levels of candidate genes in biotic stress and jasmonic acid (JA) signaling pathways using qRT-PCR, through which we discovered a unique preferential defense response of H. parviporum-infected Norway spruce under drought stress at the molecular level. Disturbances related to water availability, especially low water conditions can have negative effects on the tree host and benefit the infection ability of the pathogens in the host. From our RNA-seq analysis, 114 differentially expressed gene regions were identified between high (+) and low (−) water groups under pathogen attack. None of these gene pathways were identified to be differentially expressed from both non-treated and mock-control treatments between high (+) and low (−) water groups. Finally, only four genes were found to be associated with drought in all treatments.

Background Adaptive processes shape the evolution of genomes and the diverse functions of different genomic regions are likely to have an impact on the trajectory and outcome of this evolution. The main underlying hypothesis of this study is that the evolution of Simple Sequence Repeats (SSRs) is correlated with the evolution of the genomic region in which they are located, resulting in differences of motif size, number of repeats, and levels of polymorphisms. These differences should be clearly detectable when analyzing the frequency and type of SSRs within the genome of a species, when studying populations within a species, and when comparing closely related sister taxa. By coupling a genome-wide SSR survey in the genome of the plant pathogenic fungus Heterobasidion irregulare with an analysis of intra- and interspecific variability of 39 SSR markers in five populations of the two sibling species H. irregulare and H. annosum, we investigated mechanisms of evolution of SSRs. Results Results showed a clear dominance of trirepeats and a selection against other repeat number, i.e. di- and tetranucleotides, both in regions inside Open Reading Frames (ORFs) and upstream 5’ untranslated region (5’UTR). Locus per locus AMOVA showed SSRs both inside ORFs and upstream 5’UTR were more conserved within species compared to SSRs in other genomic regions, suggesting their evolution is constrained by the functions of the regions they are in. Principal coordinates analysis (PCoA) indicated that even if SSRs inside ORFs were less polymorphic than those in intergenic regions, they were more powerful in differentiating species. These findings indicate SSRs evolution undergoes a directional selection pressure comparable to that of the ORFs they interrupt and to that of regions involved in regulatory functions. Conclusions Our work linked the variation and the type of SSRs with regions upstream 5’UTR, putatively harbouring regulatory elements, and shows that the evolution of SSRs might be affected by their location in the genome. Additionally, this study provides a first glimpse on a possible molecular basis for fast adaptation to the environment mediated by SSRs. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2274-x) contains supplementary material, which is available to authorized users.

Volatile organic compounds (VOCs) play an important role in the communication among organisms, including plants, beneficial or pathogenic microbes, and pests. In vitro, we observed that the growth of seven out of eight Basidiomycete species tested was inhibited by the VOCs of the biocontrol agent Pseudomonas protegens strain CHA0. In the Ascomycota phylum, only some species were sensitive (e.g., Sclerotinia sclerotiorum, Botrytis cinerea, etc.) but others were resistant (e.g., Fusarium oxysporum f. sp. cubense, Verticillium dahliae, etc.). We further discovered that CHA0 as well as other ten beneficial or phytopathogenic bacterial strains were all able to inhibit Heterobasidion abietinum, which was used in this research as a model species. Moreover, such an inhibition occurred only when bacteria grew on media containing digested proteins like peptone or tryptone (e.g., Luria-Bertani agar or LBA). Also, the inhibition co-occurred with a pH increase of the agar medium where the fungus grew. Therefore, biogenic ammonia originating from protein degradation by bacteria was hypothesized to play a major role in fungus inhibition. Indeed, when tested as a synthetic compound, it was highly toxic to H. abietinum (effective concentration 50% or EC50 = 1.18 M; minimum inhibitory concentration or MIC = 2.14 M). Using gas chromatography coupled to mass spectrometry (GC/MS), eight VOCs were found specifically emitted by CHA0 grown on LBA compared to the bacterium grown on potato dextrose agar (PDA). Among them, two compounds were even more toxic than ammonia against H. abietinum: dimethyl trisulfide had EC50 = 0.02 M and MIC = 0.2 M, and 2-ethylhexanol had EC50 = 0.33 M and MIC = 0.77 M. The fungus growth inhibition was the result of severe cellular and sub-cellular alterations of hyphae occurring as early as 15 min of exposure to VOCs, as evidenced by transmission and scanning electron microscopy observations. Transcriptome reprogramming of H. abietinum induced by CHA0’s VOCs pointed out that detrimental effects occurred on ribosomes and protein synthesis while the cells tried to react by activating defense mechanisms, which required a lot of energy diverted from the growth and development (fitness cost).