Ecological constraints limit the fitness of fungal hybrids in the Heterobasidion annosum species complex

The ability of two closely related species to maintain species boundaries in spite of retained interfertility between them is a documented driving force of speciation. Experimental evidence to support possible interspecific postzygotic isolation mechanisms for organisms belonging to the kingdom Fungi is still missing. Here we report on the outcome of a series of controlled comparative inoculation experiments of parental wild genotypes and F(1) hybrid genotypes between closely related and interfertile taxa within the Heterobasidion annosum fungal species complex. Results indicated that these fungal hybrids are not genetically unfit but can fare as well as parental genotypes when inoculated on substrates favorable to both parents. However, when placed in substrates favoring one of the parents, hybrids are less competitive than the parental genotypes specialized on that substrate. Furthermore, in some but not all fungus x plant combinations, a clear asymmetry in fitness was observed between hybrids carrying identical nuclear genomes but different cytoplasms. This work provides some of the first experimental evidence of ecologically driven postzygotic reinforcement of isolation between closely related fungal species characterized by marked host specificity. Host specialization is one of the most striking traits of a large number of symbiotic and parasitic fungi; thus, we suggest the ecological mechanism proven here to reinforce isolation among Heterobasidion spp. may be generally valid for host-specialized fungi. The validity of this generalization is supported by the low number of known fungal hybrids and by their distinctive feature of being found in substrates different from those colonized by parental species.     

Transmission of double-stranded RNA in Heterobasidion annosum

Transmission of dsRNA viruses between homo- and heterokaryotic mycelia paired on agar plates and into conidia has been studied in Heterobasidion annosum. Horizontal transmission of dsRNA occurred between both homo- and heterokaryotic isolates, as well as between isolates belonging to different intersterility groups. The proportions of vertical transmission into conidia were 3% and 55%, respectively, for the two isolates included in the study. RT-PCR of dsRNA and PCR-RFLP of mitochondrial markers were used to confirm transmission of dsRNA between the cytoplasms of different mycelia. The identity of nuclei and nuclear migration during experiments were verified using PCR-RFLP of several nuclear markers.     

Production of Metal‐Chelating Compounds by Species of Heterobasidion annosum sensu lato

The role of iron and compounds that chelate iron in the development of fungal diseases and wood degradation is not well understood, and their involvement in the simultaneous pathogenic and wood‐decomposing capabilities of Heterobasidion annosum s.l. is unknown. In the current study, the production of low‐molecular‐mass compounds that can chelate iron, such as catecholate, hydroxamate and oxalate, by H. annosum s.l. was correlated positively with supplementation of the medium with iron. In contrast, iron supplementation did not increase the Fe³⁺‐reducing ability of H. annosum s.s. and H. abietinum hyphae. Indeed, H. annosum s.s. is known to cause higher mortality of the plant host, but produced a lower quantity of siderophores than H. abietinum or H. parviporum. Under iron supplementation, siderophore production was correlated with phenoloxidase activity in the low‐molecular‐mass fraction, which might have consequences for cell wall decomposition.     

The alien invasive forest pathogen Heterobasidion irregulare is replacing the native Heterobasidion annosum

Biological Invasions - Invasions by alien pathogens are a major threat to forest conservation. The North American fungal pathogen of conifers Heterobasidion irregulare, inadvertently introduced in...     

Resin Production and Susceptibility to Heterobasidion annosum in Corsican Pine

The vertical resin ducts in Corsican pine sapwood made the majorcontribution to resin yields as assessed by sampling tubes.Resin yields from roots appeared to be influenced by soil physicalfactors which affect vertical duct frequency. Yields from stemswere not affected by soil pH, as had been suggested previously,and the greater resistance of pines to Heterobasidion annosumattack on acidic soils does not seem to be due to an effectof soil pH on the resin response. Considerations of the infectionhabit suggested that the tube sampling method did not measurethe most relevant parameters of resin production in the contextof host resistance.     

Callose Synthase in Pinus sylvestris Response during Infection by Species of Heterobasidion annosum sensu lato with Varied Host Preferences

Strengthening of plant cell walls at the site of fungal entry is one of the earliest plant responses to fungal pathogens. The aim of our study was to characterize the pattern of callose synthase localization and callose deposition in roots of Pinus sylvestris after infection by species of the Heterobasidion annosum s.l. complex with different host specificity: H. annosum s.s., H. parviporum and H. abietinum. To address this, sense‐labelled probes and ribonuclease‐treated samples were used to determine in situ hybridizations of callose synthase by FISH method. Furthermore, determination of callose accumulation within P. sylvestris cells was carried out using aniline blue. The different species of H. annosum s.l. had distinct impacts on the callose synthase staining within plant tissues. Moreover, while inoculation with strains of H. abietinum resulted in callose synthase accumulation at the point of hyphae contact with the host cell, this was not observed with the other species. A significant difference in callose synthesis localization was observed after inoculation with varied species of H. annosum s.l. as a result of the specific interactions with the host.     

A novel putative partitivirus of the saprotrophic fungus Heterobasidion ecrustosum infects pathogenic species of the Heterobasidion annosum complex

We characterized the bisegmented genome of a putative double-stranded (ds) RNA virus from a Chinese isolate of the fungus Heterobasidion ecrustosum, a member of the Heterobasidion insulare species complex. The larger genomic segment of 1885bp encoded a putative RNA dependent RNA polymerase (RdRp, 585aa), and the smaller one for a putative coat protein of 521aa (1826bp). Phylogenetic analyses suggest that this novel virus species, named as 'Heterobasidion RNA virus 3 from H. ecrustosum, strain 1' (HetRV3-ec1), can be assigned to the family Partitiviridae, being most similar to the Helicobasidium mompa dsRNA mycovirus with RdRp amino acid similarity of 54%. The similarity to known viruses of other Heterobasidion species was notably low (25-39%). The virus could be experimentally transmitted to members of the Heterobasidion annosum complex: the European Heterobasidion abietinum and North American Heterobasidion occidentale, and the original host strain could be cured from the virus by thermal treatment. Microscopical observations showed that hyphae of H. ecrustosum anastomosed occasionally with H. abietinum and H. occidentale, and suggested a possible route for horizontal transmission between these sexually incompatible species. The virus infection seemed to cause variable effects on the growth rate of its fungal hosts, but the results were strongly dependent on fungal strain, growth medium and incubation temperature.     

Nuclear reassortment between vegetative mycelia in natural populations of the basidiomycete Heterobasidion annosum

Somatic incompatibility is not an absolute block to nuclear exchange between incompatible mycelia of the basidiomycete Heterobasidion annosum in vitro. Under laboratory conditions new heterokaryotic genotypes can be isolated from the gap between incompatible heterokaryons, and nuclear migration between pairs of heterokaryons grown in Petri dishes has been observed. In this study, we test the hypothesis of nuclear transfer and reassortment between heterokaryotic mycelia in natural populations of H. annosum. We developed six microsatellite markers to genotype nuclei populating 21 somatically incompatible mycelia of H. annosum isolated from a single stump of Picea abies, and found that the detected heterokaryons share nuclei; 10 of the nuclear haplotypes were found in more than one mycelium. In one isolate, four nuclear types were found in the same mycelium. These findings indicate that new heterokaryons can be formed as a result of nuclear reassortment between incompatible heterokaryotic mycelia in nature.     

Evolutionary history of the conifer root rot fungus Heterobasidion annosum sensu lato

We investigated two hypotheses for the origin of the root rot fungus Heterobasidion annosum species complex: (i) that geology has been an important factor for the speciation (ii) that co-evolutionary processes with the hosts drove the divergence of the pathogen species. The H. annosum species complex consists of five species: three occur in Europe, H. annosum s.s., Heterobasidion parviporum and Heterobasidion abietinum, and two in North America, Heterobasidion irregulare and Heterobasidion occidentale; all with different but partially overlapping host preferences. The evolution of the H. annosum species complex was studied using six partially sequenced genes, between 10 and 30 individuals of each species were analysed. Neighbour-joining trees were constructed for each gene, and a Bayesian tree was built for the combined data set. In addition, haplotype networks were constructed to illustrate the species relationships. For three of the genes, H. parviporum and H. abietinum share haplotypes supporting recent divergence and/or possible gene flow. We propose that the H. annosum species complex originated in Laurasia and that the H. annosum s.s./H. irregulare and H. parviporum/H. abietinum/H. occidentale ancestral species emerged between 45 and 60 Ma in the Palaearctic, well after the radiation of the host genera. Our data imply that H. irregulare and H. occidentale were colonizing North America via different routes. In conclusion, plate tectonics are likely to have been the main factor influencing Heterobasidion speciation and biogeography.     

Spread of Heterobasidion annosum s.s. and Heterobasidion parviporum in Picea abies 15 years after stump inoculation

The tree pathogenic fungi Heterobasidion annosum s.s. and Heterobasidion parviporum cause root and butt rot in Norway spruce (Picea abies) and produce serious economic losses to the forest sector in Europe. We experimentally studied inter- and intraspecific differences between H. parviporum and H. annosum s.s. in the way they infect stumps and spread into neighbouring trees. Eleven H. parviporum and nine H. annosum s.s. isolates were artificially inoculated on stumps of two spruce stands after first thinning. After 15 years, the same isolates were reisolated from neighbouring trees. Heterobasidion parviporum spread more frequently from the inoculated stumps to the neighbouring trees than H. annosum s.s. The surroundings of H. annosum s.s. stumps that did not spread were often colonized by H. parviporum. Heterobasidion annosum s.s. spread was restricted mainly to the areas of the plot where no other Heterobasidion genotypes had been inoculated. In such cases, H. annosum s.s. tended to develop into bigger genets than H. parviporum. The probability of stump-to-tree spread of H. parviporum depended on the diameter of the stumps, suggesting that H. parviporum spread may relate to the presence of heartwood. Both H. parviporum and H. annosum s.s. proved to be strong pathogens on Norway spruce; however, when competing for the same trees, H. parviporum seemed capable of excluding H. annosum s.s. from the stand.     

Modelling the mechanisms behind the key epidemiological processes of the conifer pathogen Heterobasidion annosum

The Heterobasidion annosum species complex is a widely distributed group of fungal conifer pathogens causing root and butt rots. We studied the key processes of Heterobasidion epidemiology by compiling models that rely on biological processes. Models were included in the mechanistic model with stochastic elements, Hmodel, simulating the fungal dynamics in even-aged Norway spruce stands. The results from the modelling and stand-level simulations indicated that primary infections are affected by the stump size and spore deposition. In addition, we found that Heterobasidion dynamics at the scale of the stand are driven by several infections in large stumps, rather than by numerous infections in small stumps. We assessed the need for quantitative results in Heterobasidion biology, especially in the spread mechanisms, to support the development of complex mechanistic models.     

Two hydrophobin genes from the conifer pathogen Heterobasidion annosum are expressed in aerial hyphae

Two hydrophobin genes (HAH1 and HAH2) have been identified in a Heterobasidion annosum infection-stage cDNA-library. Comparisons of their nucleotide and amino acid sequences show similarity to the coh1 hydrophobin from Coprinopsis cinerea and the sc3 hydrophobin from Schizophyllum commune. Both HAH1 and HAH2 display the amino acid consensus pattern of class I hydrophobins, including the spacing of eight conserved cysteine residues. Real-time quantitative RT-PCR showed high expression of both genes in aerial hyphae but low expression in submerged hyphae and during in vitro infection of pine seedlings. Segregation analysis of HAH1 and HAH2 in a defined cross of Heterobasidion annosum localised HAH1 to linkage group 3 but did not positioned HAH2 in the genetic linkage map. Sequence characteristics and expression patterns of HAH1 and HAH2 suggest a role in aerial growth of mycelia, but not during pathogenesis.     

An AFLP-markers based genetic linkage map of Heterobasidion annosum locating intersterility genes

A genetic linkage map of the basidiomycete Heterobasidion annosum, casual agent of root rot in conifers, was constructed from a compatible mating between isolates from the North American S and P intersterility groups. In a population consisting of 102 progeny isolates, 358 AFLP markers were scored. The linkage analysis generated 19 large linkage groups, containing 6 or more markers, which covered 1468 cM. The physical size to genetic distance was approximately 11.1 kbp/cM. Segregation of three intersterility gene loci were analysed through mating of the progeny isolates with three tester strains carrying known intersterility genotypes. The loci for the two intersterility genes (S and P) were successfully located in the map. Segregation of the mating type locus was analysed by backcrossing the progeny isolates with their parental strains. The mating type locus could not be located in the map.