Presence of double‐stranded RNA in Heterobasidion annosum

A search for double‐stranded RNA (dsRNA) was conducted among 204 European isolates of the pathogenic fungus Heterobasidion annosum. Nucleic acids were extracted and purified by cellulose CF‐11 chromatography or lithium chloride precipitation. dsRNA was present in eight of the isolates and was confirmed by nuclease digestion. The dsRNA elements ranged between 1.8 and 2.4 kbp and were found in two H. annosum intersterility groups, S and P. Partial amino acid sequence information from one dsRNA element showed significant homology to RNA‐dependent RNA polymerases from several fungal partitiviruses. This is the first report of the presence of dsRNAs in H. annosum. Possible implications of dsRNA for the biology of the fungus and the potential for biological control are discussed.     

Variation in Heterobasidion annosum detected by Random Amplified Polymorphic DNAs

The production of Random Amplified Polymorphic DNAs (RAPDs) by Polymerase Chain Reaction (PCR) was used to detect variation in the isolates of Heterobasidion annosum with various geographical origins. Specific RAPD products were detected for each of the intersterility groups: the European S, F, P and the North American S group. There was considerably more polymorphism found among European S than P isolates. The analysis of RAPDs was shown to be a simple and a fast way to generate DNA markers specific to the previously established intersterility groups and thus useful for diagnostic purposes.     

Variation in growth of Heterobasidion annosum among clones of Picea abies incubated for different periods of time

Thirty‐seven 4‐year‐old clones of Picea abies were inoculated with one isolate of the S intersterility group of Heterobasidion annosum in a greenhouse. The dehardened cuttings were organized in three different groups with four to six ramets in each group. All groups were inoculated on the same day shortly after shoot elongation. The groups were sampled for H. annosum growth after 34 (group 1), 83 (group 2) and 182 days (group 3), respectively. Measured parameters were cutting height and diameter, vigour index of the cuttings, infection incidence, mortality rate and fungal growth in the stem. The height of the cuttings was almost constant during the 6 months of incubation, whereas the diameter increased by about 10% during the same period. The proportion of living cuttings containing H. annosum decreased with time (99.5, 93 and 67% infection in groups 1, 2 and 3, respectively) and differed significantly among clones in group 3. Mortality rate increased with time (0.5, 22 and 37% mortality in groups 1, 2 and 3, respectively) and differed significantly among clones in groups 2 and 3; the same clones being most susceptible for both times. Mean fungal growth into the wood was significantly different among groups and among clones within each group. The ranking position for fungal growth was similar in the three groups. The results indicate that frequency of infection success, mortality rate and fungal growth are clone‐dependent factors. Broad sense heritability varied between 0.08 and 0.25 for fungal growth and lesion length at the three incubation periods. Infection success frequency was initially not different among clones but in the longer incubation periods there were significant differences among clones, indicating differences in resistance. Fungal growth in wood differed among separate host clones irrespectively of the length of inoculation period. The vigour of the cuttings seem to influence the length of fungal extension only in the initial stage of the infection.     

Fast methods for DNA extraction for PCR analyses from Heterobasidion annosum mycelium and Endocronartium pini aeciospores

The extraction of DNA from mycelium of Heterobasidion annosum or aeciospores of Endocronartium pini could be done rapidly by homogenizing the fungal material in PCR-compatible buffer in Eppendorf tubes with a disposable pestle mounted on an electric drill. The DNA yield with this method was relatively low but sufficient for PCR analysis with ITS-specific primers. For large series of fungal samples this protocol provides a quick and cheap method of determining the intersterility groups of H. annosum or for specific primer studies on E. pini populations. The time needed for DNA isolation from E. pini aeciospores and H. annosum mycelium could be reduced by nearly 85% compared to the mortar-and-pestle protocol. By using a disposable pestle mounted on an electric drill and phenol-chloroform as extraction solution, the yield of DNA was higher than with the mortar-and-pestle method with phenol-chloroform as extraction solution. The quality of DNA was sufficient for both specific-primer and RAPD analyses, but the more complicated extraction method with phenol was needed.     

Spore deposition of Heterobasidion annosum coll. in Picea abies stands of North Karelia,eastern Finland

Spore deposition of Heterobasidion annosum coll.was measured with the wood disc method in 20 Norway spruce stands in June, July and September 1999 in North Karelia, eastern Finland. The study area crossed the presently known northern boundary of the distribution area of H. annosum (62°45′N). The spore deposition rate decreased northwards and in the northernmost parts of the study area, deposition was detected only in September. On average 71% of the spore deposition consisted of intersterility group P and 29% of intersterility group S. The proportion of S‐type in the deposition increased northwards. Most of the isolated colonies were homokaryons, but the proportion of heterokaryons increased with increasing deposition rate.     

Distribution of Heterobasidion annosum intersterility groups in Poland

Specimens of Heterobasidion annosum were collected in 104 different stands in 43 regions of Poland. Pure cultures originating from 439 collections were identified in mating tests. Three intersterility groups, P, S and F, of H. annosum were found. Their occurrence in Poland was connected with the natural distribution of the main hosts: Pinus sylvestris, Picea abies and Abies alba, respectively. P was the most common intersterility group of H. annosum in Poland, causing mortality in Scots pine plantations and root rot in older stands. It was also isolated from Betula pendula, P. abies, Larix decidua, Fagus sylvatica and Carpinus betulus. The S group was present in the southern and north‐eastern parts of the country, causing root and butt rot mostly in spruce stands. The F group occurred in the south of Poland, in the mountains, highlands and lowland up to the northern border of the distribution of fir. It was found only on stumps, old dead trees and logs. There was no evidence of damage caused by the F group on A. alba trees.     

Isozyme and RAPD polymorphisms in Heterobasidion annosum in Italy

Isozyme and random amplified polymorphic DNA (RAPD) polymorphisms were used to study variability in a group of 41 isolates from the Italian population of Heterobasidion annosum. The isolates belonged to the intersterility groups P and S, and particularly to the group that is most widely distributed in Italy, group F. Isozyme analysis was effective in identifying the three intersterility groups and revealed a high degree of genetic divergence within the P group isolates; the mannose phosphate isomerase (MPI-2) locus was diagnostic in the attribution of isolates to the more correlated F and S groups. RAPDs were detected following amplification by the polymerase chain reaction (PCR). 74 RAPD fragments, obtained through amplifications with eight primers, were scored. Isolates from the 3 intersterility groups were clearly divergent based on analysis of RAPD markers. However, a similarity index calculated for the isolates within the F population indicated a high uniformity of the isolates collected throughout the Italian peninsula.     

In vitro interactions between bacteria isolated from Sitka spruce stumps and Heterobasidion annosum

Culture medium composition affected antagonism by bacterial isolates from Sitka spruce (Picea sitchensis) stumps against Heterobasidion annosum. Fifty percent of bacterial isolates inhibited H. annosum growth on sporulation agar or yeast–dextrose–peptone agar; only 10% of isolates caused inhibition on both media. Proportions of isolates inhibiting H. annosum varied with stump age; fewer isolates from 4‐ or 6‐year‐old stumps exhibited antagonism than isolates from older or younger stumps. Fifteen isolates showing antagonism on sporulation agar were tested against H. annosum in spruce wood cubes. None of the bacterial isolates alone caused a significant weight reduction in inoculated cubes. Relative inoculation times of bacterial isolates and H. annosum had an effect on weight loss in interactions; simultaneous inoculation with isolates and H. annosum inhibited weight loss caused by H. annosum compared with bacteria‐free controls. Inoculation with bacterial isolates 10 days before H. annosum had no effect on the decay rate. In contrast, inoculation with H. annosum 10 days before bacteria increased weight loss of cubes by 200% relative to cultures lacking bacteria. The effect of a mixed bacterial inoculum on weight change in 0.2‐mm spruce wood slips co‐inoculated with H. annosum, Resinicium bicolor, Hypholoma fasciculare, Stereum sanguinolentum or Melanotus proteus differed between different fungi.     

Intersterility groups of Heterobasidion annosum and their host specificity in Bulgaria

The occurrence of 46 strains of Heterobasidion annosum from four forest areas in Bulgaria is reported. Three intersterility groups, P, S and F, were identified for the first time in populations of this pathogen in Bulgaria. A strict host specificity of different intersterility groups was observed even in mixed stands. The P group was isolated from Pinus sylvestris and Pinus nigra and occasionally from stumps of Abies alba. It was rare in artificial pine plantations on dry sites, whereas it was more common on more moist sites. The F group caused severe damage on Abies alba but was not found, even as a saprotroph, on admixed pines. This is the first report of the F group on Abies alba in the Balkans. The S group was found only on Picea abies.     

Differences in virulence of genets of Heterobasidion annosum and susceptibility of young plants of different conifer species and origins

Heterobasidion species are the most important pathogens causing root and stem rot on conifers in northern hemisphere forests. The host list of this complex is very wide and includes over 200 species of trees and shrubs. Among the members of this complex, Heterobasidion annosum s. s. has the largest host range. In this study, young plants of Pinus sylvestris, Picea orientalis, Abies nordmanniana, Cedrus libani and Pinus brutia (three different origins) were inoculated on the lower stem with known genets of Heterobasidion annosum s.s. collected from Pinus brutia stands in south-western Türkiye. Infection frequency, assessed as presence of the conidial stage in stem discs following incubation, in the inoculated seedlings was 100%. The Heterobasidion annosum s. s. isolates were re-isolated from all inoculated host species. Control seedlings showed no symptoms of disease. Mortality in inoculated plants was 11.5% of the 735 inoculated plants, which died over an 8-weeks incubation period. The isolates showed greater growth on Cedrus libani, Pinus sylvestris and Picea orientalis seedlings compared to other species tested. On the other hand, it was found that the least affected seedlings were Pinus brutia TB12 and Abies nordmanniana. This study proved that differences occur in aggressiveness of Heterobasidion annosum s. s. to host species. A striking point in the results is that, despite being the host species from which the isolates were obtained, Pinus brutia seedlings showed lower sensitivity to Heterobasidion annosum s. s. than the other conifer species tested. Inoculations of three different Pinus brutia provenances suggested there was no significant difference in mean lesion lengths and fungal growth values in Pinus brutia plants, except in Pinus brutia TB14, which was more susceptible to extension growth of the pathogen.     

Intraspecific variation in Heterobasidion annosum for growth in sapwood of Picea abies and Pinus sylvestris

Two greenhouse experiments were conducted to study intraspecific variation in growth of the root rot fungus Heterobasidion annosum in living host sapwood. In experiment 1, Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings were inoculated with H. annosum isolates, 14 each of the S-and P-intersterility groups, collected from various parts of Sweden. In pine, the P-group isolates were more virulent than the S-group isolates both in terms of infection frequency, induced mortality rate (p < 0.05), and fungal growth in sapwood (p < 0.05). In spruce, the P-group isolates were also more virulent on average, but the difference was not statistically significant. Both S and P isolates had a higher infection frequency and a significantly longer sapwood growth on spruce than on pine. The P-group caused higher mortality on pine than on spruce. The length of the lesion in the inner bark was strongly correlated with fungal growth in spruce, but not in pine where the lesions were short or absent. In experiment 2, ten Norway spruce clones were inoculated with 18 S-isolates, originating from nine live-decayed trees and from nine spore-infected stumps in a single Norway spruce stand. The objective was to test whether any selection for growth rate in sapwood was detectable among individuals of H. annosum originating either from stumps or trees. The results gave no support for such selection since no difference in sapwood growth between the two groups of isolates was found.     

Swiss stone pine trees and spruce stumps represent an important habitat for Heterobasidion spp. in subalpine forests

In the Western Italian Alps (WIA), the three European species of the forest pathogen Heterobasidion spp. can coexist in the same area. Heterobasidion parviporum Niemelä & Korhonen and Heterobasidion abietinum Niemelä & Korhonen are normally found in areas with a significant presence of their respective primary hosts, spruce (Picea spp.) and fir (Abies spp.). The host/niche occupied by Heterobasidion annosum (Fr.) Bref. in the region still remains unclear. Although Scots pine (Pinus sylvestris), a major host for this fungal species in other parts of Europe, is abundant in the region, little or no evidence of disease caused by H. annosum is visible in this tree species. Two different, but not mutually exclusive, hypotheses can explain the presence of H. annosum: (1) Scots pines are infected but largely asymptomatic and (2) H. annosum has adapted to different hosts. An analysis of Heterobasidion species was performed in two natural, mixed‐conifer forests using traditional isolation techniques and novel direct molecular diagnosis from wood. In a subalpine stand of mixed spruce (Picea abies), larch (Larix spp.), and Swiss stone pine (Pinus cembra), 18 naturally infected spruces and larches only yielded H. parviporum. A Swiss stone pine in the same stand was extensively colonized by both H. parviporum and H. annosum. In a second subalpine stand, an analysis of 18 spruce stumps and nine Swiss stone pine stumps yielded both H. parviporum and H. annosum isolates. Pine stumps had been mostly colonized by H. parviporum prior to tree felling, suggesting that this species may be secondarily infected by the locally predominant Heterobasidion species (i.e. H. parviporum). Results of our analysis also indicated that primary colonization of spruce stumps (e.g. through basidiospores) was caused by both H. parviporum and H. annosum, while secondary infection of such stumps was mostly because of H. parviporum.     

In vitro inhibition of Heterobasidion annosum by Phaeotheca dimorphospora

The antagonistic fungus Phaeotheca dimorphospora has been tested in vitro on agar plates and wood discs to evaluate its potential use against P (Eastern Canada) and S (Finland) intersterility groups strains of Heterobasidion annosum using two methods. Production of antifungal metabolites by P. dimorphospora was demonstrated on agar plates as well as on Pinus resinosa and Picea abies discs using the bi‐layer technique. Diffusible metabolites inhibited the growth of the pathogen on both substrates. Direct confrontation on wood discs showed that a 7‐day period of advanced growth by P. dimorphospora completely prevented the colonization of the tissue by H. annosum. Control was partial when the antagonist had only one day of advanced growth. Growth inhibition was superior against S group strains with less than 1% of the samples colonized by H. annosum compared with 22% for P group strains. Moreover, P. dimorphospora was isolated in more than 83% of the samples in the absence of the pathogen in both wood species. In vitro tests demonstrated that P. dimorphospora is able to colonize red pine as well as Norway spruce tissue and to use wood components for the production of antifungal metabolites.     

F group of Heterobasidion annosum found in Poland

The F intersterility group of Heterobasidion annosum, specialized to Abies species and earlier reported from southern parts of Europe, was identified from Abies alba and Picea abies in the Carpathian Mountains in southern Poland, close to northern limit of the natural distribution of A. alba. However, the fungus is not common in this area and seems to be primarily a saprophyte, growing on stumps and dead trees.     

Induced resistance of Norway spruce,variation of phenolic compounds and their effects on fungal pathogens

Three clones of Norway spruce (Picea abies) were studied for their response to mass‐inoculation with the blue‐stain fungus Ceratocystis polonica. The effect of different pretreatments (fungal inoculation and wounding) before mass‐inoculation was investigated for their possible role in an acquired resistance reaction. Pretreated trees showed enhanced resistance to the subsequent mass‐inoculation relative to control trees that received no pretreatment. Furthermore, the fungal colonization of inoculated trees was less than that of wounded trees. The phenolic content of the bark, analysed by RP‐HPLC, was compared in trees receiving different treatments. Trees inoculated with C. polonica had higher average concentration of (+)‐catechin, taxifolin and trans‐resveratrol than wounded trees. Both inoculated and wounded trees had higher average concentrations of these compounds than control trees. The effect of the phenolic extract of Norway spruce bark on the growth of the root rot fungus Heterobasidion annosum and the blue‐stain fungi C. polonica and Ophiostoma penicillatum were investigated in vitro. Heterobasidion annosum was not negatively affected, and the extracts had fungistatic effects on the blue‐stain fungi. The growth of O. penicillatum was more inhibited than the growth of the more aggressive C. polonica.     

Detection and partial characterization of extracellular proteases of the pathogenic fungi Endocronartium pint,Gremmeniella abietina and Heterobasidion annosum

Protease activities from 10 homokaryotic isolates of Heterobasidion annosum., two isolates of Grem-meniella abietina and six isolates of Endocronartium pini were studied. All the fungi showed in vitro protease activity with denatured casein. The greatest caseinolytic activity was at acidic pH, but Heterobasidion annosum and Gremmeniella abietina showed caseinolytic activity at basic pH also. Protease preparations from Heterobasidion annosum were able to degrade proteins from total phloem extracts of Pinus sylvestris, Picea abies, Betula pendula and Juniperus communis. At basic pH the artificial substrates hippuryl arginine (HA) and hippuryl phenylactic acid (HPLA), were hydrolysed most rapidly by Heterobasidion annosum at pH 8.1, indicating high exopeptidase activity at this pH. According to inhibitor studies with EDTA (ethylenediaminetetraacetic acid), E64 (L-trans-epoxy-succinyl-leycylamide-(4-guanidino)-butane-agmatine) and pin2 (potato trypsin/chymotrypsin inhibi-tor) both cysteine and serine proteases were present in proteases secreted by these pathogens, although only very low protease activity at basic pH was detected with Endocronartium pini.     

Untersuchungen zur biologischen Bekämpfung von Heterobasidion annosum an Fichte (Picea abies) mit antagonistischen Pilzen

Investigations on biological control of Heterobasidion annosum in Norway spruce (Picea abies H. Karst.) with antagonistic fungi. I. Production of inoculum and interaction experiments in vitro. Thirty isolates of 22 fungal species were examined in vitro for sporulation, for germination of the spores, and for antagonism of mycelia against three strains of H. annosum. Viable spores were produced by 17 isolates, antagonism was shown by 28 isolates.     

Intersterilitätsgruppen und Klone von Heterobasidion annosum-Isolaten aus Koniferen- Wurzel- und -Stammfäulen

Intersterility groups and clones of Heterobasidion annosum isolates from root and butt rots of conifers 69 H. annousum heterokaryons from scots pine, Douglas fir, Norway spruce and larch belonged to the P group, three heterokaryons from Norway spruce to the S group. The results of a study with clones of H. annosum isolates from roots of Scotch pine trees in close neighbourhood suggest colonisation by H. annosum (partly) via root contacts.     

Root and butt rot of Todo fir (Abies sachalinensis) caused by Heterobasidion annosum s.l. in Hokkaido,Japan

The occurrence and symptoms of root and butt rot were examined in a 35 × 30 m plot of 68‐year‐old Todo fir plantation in Hokkaido, Japan. Forty‐seven percent of the cut stumps were decayed and 52% of the decayed stumps showed similar decay characteristics with yellowish orange to light brown colouration and expanded pockets in the heartwood. Morphological characteristics of the pure cultures isolated from the decay were similar to the cultures isolated from basidiocarps of Heterobasidion annosum sensu lato, found on fallen logs outside of the research site. Also DNA analysis based on the combined data set of three gene loci (glyceraldehyde 3‐phosphate dehydrogenase, heat shock protein 80–1 and elongation factor 1‐alpha genes) showed that the isolates from the decay are included in the same clade with the Japanese H. annosum s.l. isolates. They form a subclade to H. parviporum (the European S group of H. annosum s.l.). This is the first report of molecular determination of H. annosum s.l. isolated from root and butt rot in a plantation in Japan.