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Phenolic compounds are essential components of the defensive chemical arsenal of conifer trees. This chemically diverse group encompasses stilbenoids, flavonoids, isoflavonoids, proanthocyanidins and lignans. All of them are produced from the common precursors, CoA esters of the phenylpropanoids: p-coumaroyl-CoA, cinnamoyl-CoA, caffeoyl-CoA and feruloyl-CoA. Two classes of enzymes, stilbene synthases and chalcone synthases, play important roles in the biosynthesis of plant phenolics, such as the conversion of the phenylpropanoid-CoA esters into stilbenoids and flavonoids, respectively. In this study, we investigated the transcriptional responses of the Scots pine ( Pinus sylvestris) genes encoding stilbene and chalcone synthases to the infection by the fungal pathogen Heterobasidion annosum. The experiments were performed on pine seedlings and mature trees. Our results show that the stilbene synthase gene is strongly induced in both age groups, but the level of induction in adult trees varied considerably among individual trees. Systemic induction in needles and hypocotyl regions was documented in young seedlings following fungal inoculation in root region. Concurrent changes in the expression of the chalcone synthase gene in response to fungal infection were much less pronounced. Our data confirm previous observations on the differential regulation of these enzymes in Scots pine and emphasize the role of stilbene synthase in the induced response to fungal infection. [ABSTRACT FROM AUTHOR]

Gene expression levels of pathogenesis-related proteins ( PR1, PR3, PR5, PR8 and PR9), cell wall modification genes ( PAL and expansin), signal transduction genes ( ACC oxidase, AOC, MAPK) and a Myb transcription factor were compared in two Hevea brasiliensis budded clones ( RRIM612 and PR107) 5 weeks after wounding and artificial inoculation with the white rot fungus, Rigidoporus microporus. PR3 class I chitinase was upregulated in RRIM612 in response to the pathogen when compared with the wounded control. PR9 class IV peroxidase was highly upregulated in PR107. PR1 and PR8 were expressed more in wounded than in inoculated clones. Genes involved in ethylene and jasmonic acid signalling pathways and phenylalanine ammonia-lyase ( PAL) were upregulated almost equally in both clones. The Myb transcription factor was upregulated in RRIM612, while MAPK was upregulated in PR107 when compared to the wounded control. The predicted expansin-like protein was upregulated 40-fold in RRIM612 when compared to the wounded control. The results demonstrate the variability in defence responses in different clones and provide the first set of defence genes expression profiles in the host-pathogen interaction of the white rot disease of rubber trees. [ABSTRACT FROM AUTHOR]

Fungal cell wall elicitors have notable effects on germination of pine seeds, defence response and many other aspects of metabolic processes. In this study, we investigated morphological, physiological and molecular response of somatic embryos of Scots pine to fungal cell wall elicitors from ectomycorrhizal fungus Suillus bovinus, pathogenic fungus Heterobasidion annosum or weak Scots pine pathogen ( Heterobasidion parviporum). Results from the embryo rooting experiment indicated that the genotypic origin of the conifer tree and the source of the elicitors affected the root development. Elicitors from either S. bovinus or H. parviporum increased the root formation but increased concentration of elicitors from H. annosum decreased the rooting and survival rate of the embryos. At the molecular level, altered transcript changes were observed among genes encoding proteins involved in cell division, cell wall modification, and stress following exposure of embryos to elicitors. Increased levels of actin and peroxidase gene expression were detected at 7 days after exposure to S. bovinus or H. parviporum elicitors. H. annosum elicitors provoked significant induction of the ethylene synthesis gene PsACS-1. Generally the gene expression patterns at early stages observed in the subset of genes investigated after treatment with H. parviporum elicitor were found to be similar to that documented for S. bovinus. In contrast, somatic embryos exposed to H. annosum elicitor had different patterns of regulation marked by delayed response, and in some cases gradual degradation and death of the embryos were observed. [ABSTRACT FROM AUTHOR]

Thirty-eight isolates of Rhizoctonia spp. were isolated from Scots pine ( Pinus sylvestris) seedlings with damping-off symptoms, originating from two forest nurseries in central-west Poland (Wronczyn and Jarocin) and from diseased seedlings grown in soil from Wronczyn nursery. Majority of these isolates (79%) had multinucleate cells and were identified as Rhizoctonia solani. The remaining isolates were recognized as binucleate Rhizoctonia spp. R. solani isolates were characterized using hyphal anastomosis and were divided into five anastomosis groups (AG). The most prevalent was AG5 (37% of isolates), followed by AG2-1 (30%) and 27% of the isolates were identified as AG4. Groups AG1-IB and AG2-2 were only represented by single isolates. The virulence recorded as mortality (in percentage) was comparatively high for binucleate and multinucleate isolates of Rhizoctonia spp. Sequence analysis of the polymerase chain reaction (PCR)-amplified internal transcribed spacer (ITS) rDNA region was used for phylogenetic analysis. The dendrogram showed that isolates were distinctly separated based on their AG types and there was no relationship between pathogenicity on Scots pine seedlings and the AG to which the isolates belong to. The results are discussed with respect to pathogenic potential of the various AG groups. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Interaction of the necrotroph Fusarium avenaceum with seedling roots of Norway spruce was studied. The results showed that Norway spruce seedlings were susceptible to the pathogen but disease progression and severity was dependent on inoculum potential. Host recognition of the pathogen marked by hypersensitive browning reactions occurred late, thus making successful resistance to cellular damage impossible. Such host resistance responses were mostly in the form of lignification, accumulation of electron dense materials and papillae formation. [ABSTRACT FROM AUTHOR]

With aid of fluorescein isothiocyanate and gold-labeled lectins, together with antibodies against chitinase and β-1,3-glucanases, the spatial distribution of chitin and glucans in hyphal walls of Pythium dimorphum, a causative agent of root die-back disease of conifer seedlings has been studied in this article. Contrary to previous reports, this study revealed that chitin, a polymer of N-acetyl-D-glucosamine, is a component of the cell wall of this oomycetous fungus. However, compared with the other amino sugar, N-acetyl-galactosamine, it is not present in significant amounts. Ultrastructural and labeling studies demonstrated that the chitin region is embedded in or masked by glucan residues.

The article examines immunocytochemical localization of pathogenesis-related proteins in roots of Norway spruce infected with Heterobasidion annosum. The occurrence and accumulation of glucanase and chitinase in seedling roots of spruce following challenge by the root-rot pathogen Heterobasidion annosum were studied. Chitinase activity increased 2-3 fold following inoculation, whereas no significant increase in the activity levels of glucanase was recorded during infection. With TEM immunogold labelling, tile enzymes were localized in protein aggregates in host tissues and in the cell walls of intercellular hyphac.

Vascular disintegration mainly of medulla rays of spruce roots is of major significance in root rot disease of spruce caused by H. 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 against H. 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. [ABSTRACT FROM AUTHOR]

Abstract Background Symbiotic ectomycorrhizal associations of fungi with forest trees play important and economically significant roles in the nutrition, growth and health of boreal forest trees, as well as in nutrient cycling. The ecology and physiology of ectomycorrhizal associations with Pinus sp are very well documented but very little is known about the molecular mechanisms behind these mutualistic interactions with gymnosperms as compared to angiosperms. Results Using a micro-array approach, the relative abundance of 2109 EST transcripts during interaction of Pinus sylvestris roots with the ectomycorrhizal fungus was profiled. The results reveal significant differential expression of a total of 236 ESTs, 96 transcripts differentially abundant after 1 day of physical contact with the fungus, 134 transcripts after 5 days and only 6 after 15 days at early stages of mantle formation on emerging lateral roots. A subset of cell wall modification and stress related genes was further assessed by quantitative reverse transcription PCR at late stages of mycorrhizal development coinciding with Hartig net formation. The results reveal down regulation of gene transcripts involved in general defence mechanism (e.g. antimicrobial peptide) as well as those involved in cell wall modification (e.g. glycine rich protein, xyloglucan endo transglycosylase). Conclusion This study constitutes the first attempt to characterize the transcriptome of the plant partner in the Pinus sylvestris – Laccaria bicolor model system. We identified 236 ESTs which are potentially important for molecular regulation of a functional symbiotic association in conifer host. The results highlight similarities with other studies based on angiosperm model systems, nevertheless some differences were found in the timing and spatial scale of gene regulation during ectomycorrhiza development in gymnosperms. The present study has identified a number of potentially important molecular events responsible for the initiation and regulation of biochemical, physiological and morphological changes during development of a fully functional symbiosis that are relevant for gymnosperm hosts.

Using Scots pine seedlings as experimental hosts, the general reactions and recognition patterns of living conifer tissues challenged in vitro with either parasitic, saprotrophic, epiphytic or mutualistic microbial species were investigated. Three distinct reaction and recognition patterns were observed. In one, a necrosis type of browning reaction occurred that restricted vascular penetration of the seedlings when inoculated with a saprotroph (Marasmius androsaceus) or a mycorrhizal-associated bacterium (Pseudomonas fluorescens). The second pattern, a kind of necrosis in which the host defence system was ultimately ineffective, was typically observed in seedlings challenged with the S or P types of the root rot fungus (Heterobasidion annosum), the fine root parasite (Fusarium avenaceum) or the saprotrophic wood decay fungus (Phlebia gigantea). The third type, described as non-recognition, was observed on seedlings showing no necrosis and was particularly common with seedlings inoculated with either mycorrhizal fungi (Suillus granulatus, Piloderma croceum) or saprotrophs (Trichoderma aureoviride, Coriolus versicolor), or an unidentified rod-shaped bacterial isolate. Since necrosis is often linked to rapid accumulation of H2O2, increases in peroxidase activity were determined. Only seedlings challenged with the S-type of H. annosum had a systemic increase in peroxidase levels of needles, shoot and roots. Similarly, only H. annosum, F. avenaceum, P. gigantea and the Pseudomonas bacterium caused a two- to four-fold increase in the peroxidase level of inoculated roots, results suggesting that a relationship exists between the degree of necrosis and the ratio of peroxidase induction. But the higher levels of peroxidase activity was not correlated with increased resistance to disease development. [ABSTRACT FROM AUTHOR]