Root Rot Fungus Heterobasidion Annosum Research Articles

Volumetric Spore Traps Are a Viable Alternative Tool for Estimating Heterobasidion Infection Risk

Prophylactic stump treatments against the conifer root rot fungus Heterobasidion annosum s.l. should take into account the airborne inoculum density of the pathogen, in order to be economically feasible. Our objective was to test if an automatic volumetric spore trap (AVST) could be used as a sensitive alternative of passive traps for quantifying Heterobasidion airspora in forest stands. The routinely used wood disc exposure method (WDE) was implemented simultaneously with AVST in five coniferous monocultures and three near-natural stands without silvicultural management in Czechia. Air sampling took place for 24-h intervals in five months of the main sporulation period. The atmospheric concentration of H. annosum s.l. spores (cHa) was determined by qPCR with generic and species-specific primers. AVST detected more target species than WDE on 77% of sampling occasions. When comparing the relative abundance of the three European H. annosum s.l. species detected by AVST and WDE, H. parviporum and H. abietinum were found to be overrepresented on wood discs. cHa in November was significantly higher than in May and June, confirming the seasonal pattern typical for temperate Europe. For an accurate and rapid estimation of Heterobasidion primary infection risk, the usage of AVST is highly recommended.

Erratum to: Distribution of Viruses Inhabiting Heterobasidion annosum in a Pine-Dominated Forest Plot in Southern Finland

We investigated the diversity and spatial distribution of viruses infecting strains of the root rot fungus Heterobasidion annosum collected from pine stumps at a heavily infected forest site. Four different partitiviruses were detected in 14 H. annosum isolates at the study site, constituting approximately 29% of all Heterobasidion isolates investigated (N = 48). Two of the viruses detected were new partitiviruses designated here as Heterobasidion partitivirus 16 (HetPV16) and HetPV20, and two were previously known partitiviruses: HetPV7 and HetPV13. The two new partitiviruses found, HetPV16-an1 and HetPV20-an1, shared ~70% RdRp nucleotide sequence identity with the alphapartitivirus Rosellinia necatrix partitivirus 2, and less than 40% identity with known viruses of Heterobasidion spp. HetPV7-an1 was closely similar to HetPV7-pa1 isolated earlier from Heterobasidion parviporum, supporting the view of conspecific virus pools in different Heterobasidion species. Three fungal isolates were found to be co-infected with two different partitivirus strains (HetPV7-an1 and HetPV13-an2 or HetPV16-an1 and HetPV20-an1). Different isolates representing each host clone had variable virus compositions, and virus strains occurring in more than one host clone showed minor sequence variations between clones.

Quantitative interactions between the biocontrol fungus Phlebiopsis gigantea, the forest pathogen Heterobasidion annosum and the fungal community inhabiting Norway spruce stumps

Protection against the root rot fungus Heterobasidion annosum s. l. is achieved by mechanically spraying stumps with spores of the antagonistic fungus Phlebiopsis gigantea, commercialized as Rotstop®. An extensive colonisation of the stump by P. gigantea is required for protection. Colonisation may depend on how the biocontrol agent interacts with the fungal community that is endophytically inhabiting the stump, as well as with other fungi establishing after the treatment is applied. In this research, we studied how the distribution and abundance of Heterobasidion and P. gigantea is modulated by the fungal community inhabiting Norway spruce (Picea abies) stumps. First thinning stumps were treated with Rotstop and/or artificially inoculated with H. parviporum under field conditions. After two months, P. gigantea or Heterobasidion distribution was measured by qPCR, and both pathogen and biocontrol abundance gradients were correlated with members of the fungal community determined by high throughput sequencing. Rotstop application decreased Heterobasidion biomass by almost 200 times, though the strongest biomass reductions were only achieved when stumps were well covered by P. gigantea. Poor Rotstop applications resulting in, for instance, a drop of 20% in terms of stump covered area by the biocontrol, were associated with a 2-fold biomass increase of the surviving Heterobasidion colonies. Unlike Heterobasidion, some fungi such as Peniophora sp. and F. pinicola did not change their relative abundance following the Rotstop treatment and were associated with areas of low P. gigantea presence. Yeast-like fungi of the genus Debaryomyces increased its relative abundance following Rotstop treatment, and were the only taxa associated with both a higher abundance of the biocontrol, and a lower abundance of the pathogen. Ours is the first study quantifying at microscale the interaction between H. parviporum and P. gigantea in stumps subjected to Rotstop treatment, showing how incomplete stump cover may result in large shifts in terms of biomass for either the pathogen or the biocontrol. Our results stress the importance of achieving a complete cover of the stump surface in practice. Debaryomyces sp. might facilitate P. gigantea colonisation of the stump and could be added as enhancers of Rotstop treatment, though the mechanism of interaction with the pathogen and the biocontrol should be further studied.

Distribution of Viruses Inhabiting Heterobasidion annosum in a Pine-Dominated Forest Plot in Southern Finland.

We investigated the diversity and spatial distribution of viruses infecting strains of the root rot fungus Heterobasidion annosum collected from pine stumps at a heavily infected forest site. Four different partitiviruses were detected in 14 H. annosum isolates at the study site, constituting approximately 29% of all Heterobasidion isolates investigated (N=48). Two of the viruses detected were new partitiviruses designated here as Heterobasidion partitivirus 16 (HetPV16) and HetPV20, and two were previously known partitiviruses: HetPV7 and HetPV13. The two new partitiviruses found, HetPV16-an1 and HetPV20-an1, shared ~70% RdRp nucleotide sequence identity with the alphapartitivirus Rosellinia necatrix partitivirus 2, and less than 40% identity with known viruses of Heterobasidion spp. HetPV7-an1 was closely similar to HetPV7-pa1 isolated earlier from Heterobasidion parviporum, supporting the view of conspecific virus pools in different Heterobasidion species. Three fungal isolates were found to be co-infected with two different partitivirus strains (HetPV7-an1 and HetPV13-an2 or HetPV16-an1 and HetPV20-an1). Different isolates representing each host clone had variable virus compositions, and virus strains occurring in more than one host clone showed minor sequence variations between clones.

Population structure of a novel putative mycovirus infecting the conifer root-rot fungus Heterobasidion annosum sensu lato

We describe a novel putative mycovirus infecting the conifer root-rot fungus Heterobasidion annosum sensu lato. This virus, designated as Heterobasidion RNA virus 6 (HetRV6), is taxonomically distant from all previously known viruses of Heterobasidion species, but somewhat related to the Curvularia thermal tolerance virus and the Fusarium graminearum virus 4. Based on a population analysis including 35 virus strains from Heterobasidion abietinum, Heterobasidion parviporum, Heterobasidion annosum sensu stricto and Heterobasidion occidentale, HetRV6 showed a considerable degree of geographical and host-related differentiation. The North American and Eurasian virus populations were clearly separated. In Eurasia, we observed cases of discrepancy between virus and host taxonomy, suggesting interspecies virus transfer. HetRV6 was also successfully transmitted between the three European species H. abietinum, H. annosum and H. parviporum. Based on growth rate tests on agar plates and spruce stem pieces, HetRV6 seemed to be cryptic or slightly mutualistic to its host.

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.

Epidemiology of Heterobasidion abietinum and Viscum album on silver fir (Abies alba) stands of the Pyrenees

SummaryIn the last two decades, stand decline and increased mortality has affected silver fir (Abies alba) forests in the Spanish Pyrenees. Simultaneously severe occurrences of the root rot fungus Heterobasidion annosum s.l. and of the mistletoe Viscum album have been reported. We aimed to improve the understanding of the epidemiology of both pathogens in our region. All H. annosum isolates found on silver fir were typed as H. abietinum. H. abietinum was more frequently observed where cuttings had targeted fir trees rather than other species. H. abietinum fruiting bodies were observed in the most recently cut stumps. V. album was more abundant on more dominant fir trees, and in southern aspect stands. The number of V. album colonies in the stand correlated (R2 = 0.40) with silver fir mortality. Stands with a high level of V. album infection tended to have a smaller percentage of basal area in species other than silver fir, and they tended to be located on more south‐facing slopes. H. abietinum was widespread in silver fir forests of the Pyrenees. Our data suggest that, in the Pyrenees, the observed H. abietinum incidence may represent a combination of both primary and secondary spread of the pathogen. Favouring mixed forests should be tested as a potential control method for V. album. The correlation between silver fir mortality and V. album infection warrants further study, as the observed tree mortality could have occurred due to other factors than V. album, such as drought damage.

WS-5995 B, an antifungal agent inducing differential gene expression in the conifer pathogen Heterobasidion annosum but not in Heterobasidion abietinum

The mycorrhization helper bacterium Streptomyces sp. AcH 505 inhibits Norway spruce root infection and colonisation by the root and butt rot fungus Heterobasidion annosum 005 but not by the congeneric strain Heterobasidion abietinum 331 because of higher sensitivity of H. annosum 005 towards the AcH 505-derived naphthoquinone antibiotic WS-5995 B. Differences in antibiotic sensitivity between two isolates belonging to two species, H. annosum 005 and H. abietinum 331, were investigated by comparative gene expression analysis using macroarrays and quantitative RT-PCR after WS-5995 B, structurally related mollisin and unrelated cycloheximide application. Treatment with 25 microM WS-5995 B for 2 h resulted in a significant up-regulation of expression of inosine-5'-monophosphate dehydrogenase, phosphoglucomutase and GTPase genes, while the expression of genes encoding for thioredoxin and glutathione dependent formaldehyde dehydrogenase was down-regulated in the sensitive fungal strain. No differential expression in the tolerant strain was detected. Application of WS-5995 B at higher concentrations over a time course experiment revealed that H. annosum 005 and H. abietinum 331 responded differently to WS-5995 B. The fungal gene expression levels depended on both the concentration of WS-5995 B and the duration of its application. The WS-5995 B-unrelated cycloheximide caused highly specific changes in patterns of gene expression. Our findings indicate considerable variations in response to bacterial metabolites by the isolates of the conifer pathogen.

Changes in host chitinase isoforms in relation to wounding and colonization by Heterobasidion annosum: early and strong defense response in 33-year-old resistant Norway spruce clone

We studied the defense reactions of 33-year-old susceptible and resistant clones of Norway spruce (Picea abies (L.) Karst.) to the major root-rot fungus Heterobasidion annosum (Fr.) Bref. and determined if tissue cultures can be used as a model system for studying defense responses of mature trees at the molecular level. Quantitative PCR analysis of genomic DNA obtained from samples taken at different times along the lesion length in living bark indicated that the fungus was present in higher amounts and extended further into the host tissue in the susceptible clone than in the resistant clone. In protein extracts from the same lesion samples, there were differences in temporal and spatial changes in host chitinase isoform profiles between the resistant and susceptible clones. Host chitinase isoforms with pI values approximately 4.8, 4.4 and 3.7 increased more during the first 7 days after wounding and inoculation and extended further along the lesion length in the resistant clone than in the susceptible clone. These results suggest that the time from wounding and infection to induction of defense-related expression is shorter in the resistant clone indicating a more efficient host defense response than in the susceptible clone. Tissue cultures from the same clones were not resistant to H. annosum and showed no difference in the timing of the increase in chitinase isoforms in response to the pathogen. However, tissue cultures from both clones showed an increase in chitinase isoforms within 6 to 24 h past inoculation, indicating that increased chitinase expression in response to the pathogen is part of a general defense response common to both mature clones and tissue cultures.

Pathogen-inducible cDNAs from the interaction of the root rot fungus Heterobasidion annosum with Scots pine ( Pinus sylvestris L.)

Subtractive hybridization was used to select cDNAs representing genes that are differentially expressed during interaction of the necrotroph Heterobasidion annosum and its conifer host ( Pinus sylvestris). We obtained 966 ESTs from the subtraction cDNA library, which included 509 singletons and 147 contigs. The sequences of 492 clones (51%) significantly matched National Centre for Biotechnology Information Database entries. Four hundred and seventy-four ESTs (49%) had not been previously described. The ESTs with moderate to high similarity scores based on BlastX were organized into categories based on their putative function. Among the genes identified, 16% were associated with metabolism and other cellular functions, 14% with cell rescue and defence and 39% were classified as unknown. Seven of the genes shared significant homology to fungal genes. A cDNA encoding an antimicrobial peptide (AMP) was the most abundant transcript representing 2% of the total sequenced clones. The expression pattern of five ESTs (peroxidase, anti-microbial peptide, resistance gene analogue, unknown protein, thaumatin) were analysed by virtual Northern blot, and confirmed elevated levels of the gene transcripts upon pathogen infection. These ESTs provide insight into the host–pathogen interaction and also represent a resource for future research on H. annosum–conifer pathosystems.

Silvicultural and pathological evaluation of Scots pine afforestations mixed with deciduous trees to reduce the infections by Heterobasidion annosum s.s.

One possible option to avoid infections of the root rot fungus Heterobasidion annosum s.s. in plantations of Pinus sylvestris is to thin as few times and as late as possible. For this purpose, experimental plantations of P. sylvestris were established on former agricultural land in central Lithuania, where pine seedlings had been sparsely planted in different mixture schemes with the deciduous species Robinia pseudoacacia, Amorpha fruticosa and Betula pendula. As a control, two plantations of pure P. sylvestris were established at the same site in different densities. We estimated tree and stand characteristics in the experimental plantations at the age of 25 years and identified the community of wood-inhabiting fungi. No infection of H. annosum s.s. was found. The results showed that intercropping allows delayed thinnings and a better economical result could be obtained in mixed than in pure pine stands. Crown width and stem taper of the individual trees, however, were negatively affected by the wide spacing, but still remaining acceptable from a silvicultural point of view. The fungal flora differed between externally healthy and declining pines within a forest site. Higher mycodiversity was revealed in wood of declining trees.

Induction of Pinus sylvestris PsACRE, a homology of Avr9/Cf-9 rapidly elicited defense-related gene following infection with root rot fungus Heterobasidion annosum

A novel defense-related gene code named PsACRE, an Avr9/ Cf- 9 rapidly elicited ( ACRE) gene homology was isolated from the conifer tree Scots pine ( Pinus sylvestris) following infection with the root rot pathogen— Heterobasidion annosum. This is the first report of such genes to be characterized in conifer species. The full length PsACRE gene sequence from the Scots pine was obtained using the rapid amplification of cDNA ends (RACE) method with the aid of gene-specific primers. The transcript of ∼1.1 kb encodes a peptide of 207 amino acids with a 5′-untranslated region of about 210 bp long and a 3′-untranslated region of about 224 bp long. The peptide encoded by the PsACRE share high homology to a gene from Nicotiana tabacum described to encode ACRE protein 146. Amino acid analyses confirmed that the PsACRE gene belongs to leucine rich repeat (LRR) gene superfamilies. Southern gel blot analysis of genomic DNA following digestion with three restriction enzymes ( BamHI, EcoRI, HindIII) indicated that several copies of the gene are present in Scots pine genome. RNA gel blot analyses showed that the PsACRE gene was highly expressed in root and stem regions compared to needle tissues. Further, Northern analyses confirmed that the PsACRE gene expression is elevated in Scots pine roots upon infection with H. annosum. The potential significance of the PsACRE gene in host–parasite interaction in conifer pathosystem is discussed.

Induction of Pinus sylvestris PsACRE, a homology of Avr9/Cf-9 rapidly elicited defense-related gene following infection with root rot fungus Heterobasidion annosum

A novel defense-related gene code named PsACRE, an Avr9/Cf-9 rapidly elicited (ACRE) gene homology was isolated from the conifer tree Scots pine (Pinus sylvestris) following infection with the root rot pathogen—Heterobasidion annosum. This is the first report of such genes to be characterized in conifer species. The full length PsACRE gene sequence from the Scots pine was obtained using the rapid amplification of cDNA ends (RACE) method with the aid of gene-specific primers. The transcript of ∼1.1 kb encodes a peptide of 207 amino acids with a 5′-untranslated region of about 210 bp long and a 3′-untranslated region of about 224 bp long. The peptide encoded by the PsACRE share high homology to a gene from Nicotiana tabacum described to encode ACRE protein 146. Amino acid analyses confirmed that the PsACRE gene belongs to leucine rich repeat (LRR) gene superfamilies. Southern gel blot analysis of genomic DNA following digestion with three restriction enzymes (BamHI, EcoRI, HindIII) indicated that several copies of the gene are present in Scots pine genome. RNA gel blot analyses showed that the PsACRE gene was highly expressed in root and stem regions compared to needle tissues. Further, Northern analyses confirmed that the PsACRE gene expression is elevated in Scots pine roots upon infection with H. annosum. The potential significance of the PsACRE gene in host–parasite interaction in conifer pathosystem is discussed.

Use of Scots pine seedling roots as an experimental model to investigate gene expression during interaction with the conifer pathogen Heterobasidion annosum (P-type).

The root-rot fungus Heterobasidion annosum is a major pathogen of woody trees in temperate regions of the world. In this study, seedling root of Scots pine was used as an experimental model to investigate gene expression in conifer trees during challenge with H. annosum. Initial cellular and histochemical studies have established the systems and indicated the key sequence of events during the infection process. Also, to correlate histochemical observations with the time-dependent pattern of events in host gene expression, a transcriptome profiling of a selected set of host genes from a pine-root subtraction cDNA library was conducted. Differential screening of the subset of genes arrayed on nylon membrane with cDNA probes made from seedling roots infected for 1, 3, 7 and 15 days revealed a number of up-regulated genes [disease-resistance gene analog, antimicrobial peptide (AMP) gene homolog etc.] following inoculation. The results also showed strong expression of genes involved in cell defense and protein synthesis at the early stages of the infection (3-7 days) with a decline at late stages of infection (15 days). The decline in expression of key defense genes at late stages of infection correlated well with the period of vascular colonization and subsequent loss of root turgor. Northern analyses with two of the major induced genes (AMP homolog and disease-resistance gene analog) indicated a several-fold increase in host gene expression following infection. In addition, a particular single gene (thaumatin-like protein) was consistently expressed throughout the four sampling periods of the experiment. BlastX analyses revealed that the Scots-pine thaumatin-like gene shared 51-77% sequence homology with other thaumatin-like proteins in GenBank. The importance of these results in tree defense and use of conifer seedling root in host-parasite interaction in forest trees is discussed.

Isolation of a novel antimicrobial peptide gene ( Sp-AMP) homologue from Pinus sylvestris (Scots pine) following infection with the root rot fungus Heterobasidion annosum

A new family of antimicrobial peptide homologues termed Sp-Amp has been discovered in Pinus sylvestris (Scots pine). This is the first report of such proteins to be characterized in a conifer species. Sp-AMP1 was identified in a substructured cDNA library of root tissue infected with the root rot fungus Heterobasidion annosum and encodes a mature peptide of 79 amino acid residues. Three additional members of the Sp-AMP family ( Sp-AMPs 2–4) encode cysteine-rich proteins of 105 amino acids, each containing an N-terminal region with a probable cleavage signal sequence. Northern analysis confirmed that Sp-AMP expression is elevated in Scots pine roots upon infection with H. annosum. These peptides share 64% amino acid identity with a mature protein from Macadamia integrifolia ( MiAMP1), which allowed us to build a homology model for preliminary analysis. Southern analyses further confirmed that several copies of the gene are present in the Scots pine genome. The potential significance of Sp-AMP in the H. annosum–conifer pathosystem is discussed.

Differential anatomical response of Norway spruce stem tissues to sterile and fungus infected inoculations

The anatomical defense responses in stems of Norway spruce (Picea abies) clones of different resistance to pathogenic fungi were characterized over time and distance from small mechanical wounds or wounds inoculated with the root rot fungus Heterobasidion annosum. Common responses for both treatments included division of ray parenchyma and other cells in the cambial zone, accumulation of phenolic inclusions in ray parenchyma cells, activation of phloem parenchyma (PP) cells, and formation of traumatic resin ducts (TDs) in the xylem. TD formation occurred synchronously from a tangential layer of cells, or symplasmic domain, within the zone of xylem mother cells. TD induction is triggered by a signal, which propagates a developmental wave in the axial direction at about 2.5 cm per day. TDs are formed at least 30 cm above single inoculations within 16–36 days after inoculation. The size and number of TDs is attenuated further away from the inoculation site, indicating a dose-dependent activity leading to TD development. Compared to sterile wounding, fungal inoculation gave rise to more and larger TDs in all clones, and multiple rows of TDs in weak clones. Fungal inoculation also induced the formation of more new PP cells, increasing the number of PP cells in the phloem in the year of inoculation up to 100%. TD and PP cell formation was greater in susceptible compared to resistant clones and after fungal versus sterile inoculation. Potential mechanisms responsible for this variable response are discussed.

Molecular markers reveal genetic isolation and phylogeography of the S and F intersterility groups of the wood-decay fungus Heterobasidion annosum

The root–rot fungus Heterobasidion annosum (Fr.) Bref. species complex consists of three intersterility groups (S, F, and P), separated by their host affinity. The phylogenetic relationship of the species complex was studied, with the focus on the S and F groups, by comparing DNA sequences of four nuclear gene fragments: calmodulin, glyceraldehyde 3-phosphate dehydrogenase, heat stress protein 80-1, and elongation factor 1-α, and one anonymous locus, from 29 fungal isolates originating from Europe, Asia, and North America. The phylogeny of each separate gene locus as well as the combined dataset consisted of three main clades: European F group isolates, Euroasian S group isolates, and North American S group isolates, suggesting them to be separated into phylogenetic species. The results also support the hypothesis of an early separation between the S and F groups, indicating that their distribution have followed their host tree species for a considerable time period.

The putative gymnosperm plant defensin polypeptide (SPI1) accumulates after seed germination, is not readily released, and the SPI1 levels are reduced in Pythium dimorphum-infected spruce roots.

The putative plant defensin SPI1 cDNA from the conifer Norway spruce (Picea abies) is the only known plant defensin-like sequence from a gymnosperm. The predicted translational product SPI1 was not detected in the embryo or other parts of the seed by means of antibodies, but it accumulated in the root cortex after germination. In roots of seedlings infected with the root pathogenic oomycete Pythium dimorphum and the blue stain fungus Ceratocystis polonica, variable levels of SPI1 was detected during the first day as a response to the infection, however a significant increase was seen as an initial response to the root-rot fungus Heterobasidion annosum. After the first day of infection, the amount of SPI1 polypeptide was dramatically reduced in response to either of the pathogens, but not in response to the ectomycorrhizal fungus Laccaria bicolor. During the same time of infection, extensive damage to cortical root cells resulted from the infecting pathogens, but not from the mycorrhiza. These results indicate that pathogens may reduce the level of SPI1 by suppressing its expression, but may also reduce the SPI1 level by invading and disrupting the root cortical cells or by a combination of these mechanisms.

Treatment of Norway spruce and Scots pine stumps with urea against the root and butt rot fungus Heterobasidion annosum—possible modes of action

The mode of action of urea in inhibiting infection of stumps of Norway spruce and Scots pine by Heterobasidion annosum was investigated. Stem discs from both species were treated with either urea, ammonium salt, ammonia or potassium hydroxide. Neither urea per se nor ammonium ions affected the growth of H. annosum, but the fungus was inactivated in wood at pH≥7. Urea treatment of pine stem discs or stumps caused a significant rise in both pH (to pH>8) and ammonia content within 24 h in sapwood but not in heartwood. In spruce, rises in pH started in the outer sapwood and were followed by sequential increases progressively towards the heartwood. After urea application to freshly-cut pine and spruce stumps, ammonia formation and pH rise started immediately, and initially there was a correlation between pH rise and ammonia content in the wood. High pH and high ammonia content were maintained for at least 3–4 months in warm humid weather. Ammonia was released to a greater extent in spruce and pine bark than in the adjacent sapwood but without a corresponding rise in pH. Studies on the urease activity suggested that the urea was initially decomposed by urease of host rather than microbial origin. The rate of ammonia decomposition and pH rise was found to be strongly dependent on temperature.

Induction of disease resistance in Norway spruce (Picea abies) by necrotizing fungi

Norway spruce trees (Picea abies) preinoculated with the root rot fungus Heterobasidion annosum, Nectria fuckeliana or a pathogenic strain of the blue‐stain fungus Ceratocystis polonica were more efficiently protected against a subsequent massive inoculation with pathogenic C. polonica than trees pretreated with nonpathogenic C. polonica or sterile malt agar. Control trees that received no pretreatment were extensively colonized by the mass inoculation. There was a strong negative correlation between the length of the phloem necroses induced by the pretreatment inoculations and the extent of host symptoms caused by mass inoculation with pathogenic C. polonica. The degree of induced resistance in Norway spruce thus depended on the amount of host tissue destroyed by the pretreatment.