The primary module in Norway spruce defence signalling against H. annosum s.l. seems to be jasmonate-mediated signalling without antagonism of salicylate-mediated signalling

A key tree species for the forest industry in Europe is Norway spruce [Picea abies (L.) Karst.]. One of its major diseases is stem and butt rot caused by Heterobasidion parviporum (Fr.) Niemelä & Korhonen, which causes extensive revenue losses every year. In this study, we investigated the parallel induction of Norway spruce genes presumably associated with salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways previously observed in response to H. parviporum. Relative gene expression levels in bark samples of genes involved in the salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways after wounding and inoculation with either the saprotrophic biocontrol fungus Phlebiopsis gigantea or with H. parviporum were analysed with quantitative PCR at the site of the wound and at two distal locations from the wound/inoculation site to evaluate their roles in the induced defence response to H. parviporum in Norway spruce. Treatment of Norway spruce seedlings with methylsalicylate, methyljasmonate and inhibitors of the jasmonic acid/ethylene signalling pathway, as well as the Phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid were conducted to determine the responsiveness of genes characteristic of the different pathways to different hormonal stimuli. The data suggest that jasmonic acid-mediated signalling plays a central role in the induction of the genes analysed in this study irrespective of their responsiveness to salicylic acid. This may suggest that jasmonic acid-mediated signalling is the prioritized module in the Norway spruce defence signalling network against H. parviporum and that there seems to be no immediate antagonism between the modules in this interaction.     

Fungal Community Shifts in Structure and Function across a Boreal Forest Fire Chronosequence

Forest fires are a common natural disturbance in forested ecosystems and have a large impact on the microbial communities in forest soils. The response of soil fungal communities to forest fire is poorly documented. Here, we investigated fungal community structure and function across a 152-year boreal forest fire chronosequence using high-throughput sequencing of the internal transcribed spacer 2 (ITS2) region and a functional gene array (GeoChip). Our results demonstrate that the boreal forest soil fungal community was most diverse soon after a fire disturbance and declined over time. The differences in the fungal communities were explained by changes in the abundance of basidiomycetes and ascomycetes. Ectomycorrhizal (ECM) fungi contributed to the increase in basidiomycete abundance over time, with the operational taxonomic units (OTUs) representing the genera Cortinarius and Piloderma dominating in abundance. Hierarchical cluster analysis by using gene signal intensity revealed that the sites with different fire histories formed separate clusters, suggesting differences in the potential to maintain essential biogeochemical soil processes. The site with the greatest biological diversity had also the most diverse genes. The genes involved in organic matter degradation in the mature forest, in which ECM fungi were the most abundant, were as common in the youngest site, in which saprotrophic fungi had a relatively higher abundance. This study provides insight into the impact of fire disturbance on soil fungal community dynamics.     

Identification and analysis of differentially expressed cDNAs during nonself-competitive interaction between Phlebiopsis gigantea and Heterobasidion parviporum

The molecular factors regulating interspecific interaction between the saprotrophic biocontrol fungus Phlebiopsis gigantea and the conifer pathogen Heterobasidion parviporum were investigated. We constructed cDNA libraries and used expressed sequence tag analysis for the identification and characterization of genes expressed during the self and nonself-hyphal interaction. cDNA clones from either the pathogen or biocontrol agent were arrayed on nylon membrane filters and differentially screened with cDNA probes made from mycelia forming the barrage zone during nonself-interactions, mycelia growing outside the barrage zones or monocultures. BlastX analysis of the differentially expressed clones led to the identification of genes with diverse functions, including those with potential as virulence factors, such as hydrophobins. Because of the high sequence conservation (r2 = 0.81) between P. gigantea and H. parviporum, a selected number of genes from either fungus were used to monitor the expression profile under varying interaction conditions by virtual northern blot. The results are discussed with respect to the potential role of the induced genes during the nonself-competitive interaction for space and nutrients between P. gigantea and H. parviporum.     

The impacts of treatment with biocontrol fungus (Phlebiopsis gigantea) on bacterial diversity in Norway spruce stumps

The biocontrol agent Phlebiopsis gigantea has been intensively applied to the surface of Picea abies stumps to control Heterobasidion root rot. But little is known about the possible impact of this treatment on the resident bacteria community in the stumps. High throughput DNA bar-coded pyrosequencing was used to characterize the diversity of bacteria in the stumps of P. abies at 1, 6 and 13 years after treatment with P. gigantea. The sequences were classified into 12 phyla and 160 genera, of which Proteobacteria and Acidobacteria were the most abundant groups over time. Moreover, at the initial stages of decay, Proteobacteria were the most abundant whereas Acidobacteria were the most common at advanced stages of decay. Treatment with P. gigantea led to significant increase of the genus Acidobacteria-Gp1 at 1 year after treatment. The analysis of observed and estimated operational taxonomic units (OTUs) as well as diversity indices revealed that P. gigantea treatment significantly decreased the initial bacterial richness in the stumps, but the bacterial community gradually recovered and the negative effects of P. gigantea was attenuated. These results provide additional insight on the risk assessment as well as environmental impact on the long-term use of P. gigantea in the control of Heterobasidion root rot in conifer forests.     

Infection and disintegration of vascular tissues of non-suberized roots of spruce byHeterobasidion annosum and use of antibodies for characterizing infection

Vascular disintegration mainly of medulla rays of spruce roots is of major significance in root rot disease of spruce caused byH. annosum. Using seedling roots as an experimental model, the possible routes and initial host reactions preceding invasion of vascular tissues was investigated. Transmission electron microscopy showed that penetration through the endodermis was an obvious route but not without host resistance. Using antibodies againstH. annosum hyphal materials, some labelling of vascular tissues remote from sites of fungal colonization suggest the release of fungal secretory products partly active in tissue disintegration. Similarly, intense labelling was also observed in severely colonized host tissues at late stages of infection. Strong labelling recorded at 3 d p.i. mainly on fungal hyphae and scant gold particles on invaded host tissues could imply that induction of host antifungal metabolites may have been a late event. A correlation was found between total antigenic material in root homogenates measured by ELISA, density of tissue labelling by immunocytochemistry and severity of disease symptoms. The importance of this in relation to diagnosis of biotic root rot diseases in the field is discussed.     

The effects of co-fungal cultures and supplementation with carbohydrate adjuncts on lignin biodegradation and substrate digestibility

The ability of three fungal strains (Pleurotus sajor-caju, Phanerochaete chrysosporium, Trametes versicolor) to decrease the lignin content and to enhance in vitro rumen digestibility of lignified spruce sawdust was assessed. In monoculture solid substrate fermentation (SSF) studies, a considerable length of time (6 weeks) elapsed before 4 to 14% lignin was degraded. In contrast, paired or multiple cultures of these fungi caused an 8 to 16% loss of native lignin within three weeks of incubation. There were also synergistic effects on total polysaccharide/hemicellulose degraded by mixed cultures. A similar observation was made for in vitro digestibility of fungal fermented samples: Total solubles (carbohydrate products) which accumulated in cultures were significantly higher in mixed cultures than in respective monocultures. In contrast, mixtures of cell free enzyme extracts of these fungi did not cause any marked reduction in lignin or cellulose content. Supplementation of wood sawdust with carbohydrate adjuncts prior to fungal treatment also led to substantial reduction in lignin content and increased substrate digestibility.F.O. Asiegbu is with the Department of Forest Mycology & Pathology, Swedish University of Agricultural Sciences, P.O. Box 7026, S-750 07 Uppsala, Sweden; A. Paterson and J.E. Smith are with the Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, G1 1XW, UK.     

Inhibition of cellulose saccharification and glycolignin-attacking enzymes of five lignocellulose-degrading fungi by ferulic acid

At 5 g/l, ferulic acid, a plant cell-wall phenolic, severely repressed growth of the lignocellulose-degrading fungi Trichoderma harzianum, Chaetomium cellulolyticum, Phanaerochaete chrysosporium, Trametes versicolor and Pleurotus sajor-caju. At 0.5 g/l, howerver, it slightly stimulated growth of the latter two organisms. Two classes of extracellular enzymes involved in cellulose and glycolignin breakdown were assayed: cellulases; and phenol oxidases as laccases. All of the strains depolymerized cellulose but two (T. versicolor and P. sajor-caju) also secreted laccases. Laccase-secreting fungal species had normal levels of cellulose saccharification except in the presence of 5 g ferulic acid/l, whereas saccharification by the other strains was suppressed at all concentrations of the phenolic tested.