Intersterility group differentiation in Heterobasidion annosum using ribosomal IGS1 region polymorphism

Summary The intergenic spacer region 1 (IGS1) of the nuclear ribosomal RNA gene repeat was examined in three European intersterility groups of the wood‐rot fungus Heterobasidion annosum. The IGS1 elements were informative for restriction fragment length polymorphism (RFLP) fingerprinting the fungal intersterility groups following digestion with two different four‐cutter restriction endonucleases. The fingerprints of isolates from the same intersterility group from different localities shared some fragments in common, but also showed specific fragments suggesting that RFLP of the IGS1 region might be used in population studies. RFLP patterns between isolates from different intersterility groups differed more markedly. RFLP were also detected in the IGS1 region of North American strains of H. annosum and in the related species Heterobasidion araucariae. The results indicate that ribosomal IGS1 region fingerprinting could be a useful tool to identify the fungal intersterility group in Heterobasidion annosum.     

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.     

Early infection by Heterobasidion annosum in Larix x eurolepis seedlings planted on infested sites

The incidence of Heterobasidion annosum was examined in three young hybrid larch (Larix × eurolepis) plantations in southern Sweden that were established after felling Norway spruce (Picea abies) stands heavily infected by the fungus. The incidence of H. annosum was 7 %, 33 %, and 70 %, respectively, in the 2-, 3-, and 5-year-old plantations. In all plantations, H. annosum was the most frequent rot-causing fungus (81 %) and all isolates tested belonged to the P-intersterility group. No visible external differences between infected and healthy seedlings were found on any site. Both tap and lateral roots were infected by H. annosum. The results suggest that when hybrid larch is planted on sandy soils after clear felling of Norway spruce heavily infected by H. annosum, it is very susceptible to infection by H. annosum. Furthermore, transfer of H. annosum from infected stumps to hybrid larch occurs during the first few years after planting on scarified sites. In this study, scarification may have exacerbated the root disease situation and actually led to more early infections by distributing pieces inoculum across the sites.     

The couch grass rhizome with Heterobasidion annosum fruiting bodies in afforested post‐agricultural land

The pathogenic fungus Heterobasidion annosum was identified in the rhizome of a couch grass (Elymus repens) using genetic markers. The couch grass grew in the proximity of a Scots pine stand that was visibly affected by the pathogen. The rDNA analysis showed 100% sequence similarity between two H. annosum sequences of ITS1 and ITS2 obtained from fruiting bodies found on the couch grass rhizome and on the stem of the Scots pine. These results indicate a close relationship between the two isolates of H. annosum and that they are derived from a common ancestor. The results show that H. annosum mycelia can infect couch grass rhizomes and subsequently produce fruiting bodies. It also suggests that the known modes of infection of tree roots by this pathogen in coniferous stands should take into account couch grass rhizomes in the dispersal of the disease in afforested post‐agricultural soils.     

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.     

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.     

Growth of Phlebiopsis gigantea in wood of seven conifer species

Heterobasidion parviporum and Heterobasidion annosum are widely distributed root‐rot fungi that infect conifers throughout Europe. Infection of conifer stumps by spores of these pathogens can be controlled by treating fresh stumps with a competing non‐pathogenic fungus, Phlebiopsis gigantea. In this study, growth of three Latvian strains of P. gigantea and the biological control agent ‘Rotstop’ strain was evaluated in stem pieces of Norway spruce, Scots pine, lodgepole pine, Douglas‐fir, Weymouth pine, Siberian larch and Sitka spruce. The growth rates of one H. parviporum and one H. annosum isolate were also measured in the same stem pieces. The growth rate of P. gigantea varied greatly in wood of different conifer species. It was higher in the three pine species, lower in Norway spruce and lowest in Sitka spruce and Siberian larch, and in Douglas‐fir, this fungus did not grow. The largest area of wood occupied by P. gigantea was in lodgepole pine. Growth of Latvian isolates of P. gigantea in the wood of Pinus and Picea species was comparable to that of the Rotstop isolate. Consequently, stump treatment with local P. gigantea isolates should be recommended. However, our results suggest that Douglas‐fir stump treatment against Heterobasidion by P. gigantea may be ineffective and other stump treatment methods should be considered.     

Field tests on biological control of Heterobasidion annosum by Phaeotheca dimorphospora in comparison with Phlebiopsis gigantea

In field tests conducted on freshly cut red pine (Pinus resinosa) logs, the antagonistic fungus Phaeotheca dimorphospora formulated as pre‐colonized rye grains reduced colonization of wood tissue by Heterobasidion annosum to less than 2% over the 2 months following inoculation. However, compared to controls, a water suspension of P. dimorphospora did not reduce the infection, but rather promoted colonization by H. annosum after a 2‐month exposure. Production of antifungal metabolites by P. dimorphospora in the rye grains and better colonization of the logs by the antagonist were observed with the rye grain formulation. In this field test, the most effective treatment to control H. annosum was the application of an oidial suspension of Phlebiopsis gigantea. This fungus completely inhibited colonization by H. annosum mostly by rapid colonization of wood tissue. All treatments with P. gigantea, P. dimorphospora and rye grains influenced the naturally occurring mycoflora. The most important species composing this mycoflora were Hormonema sp., Mortierella spp., Mucor hiemalis f. hiemalis, Nectria sp., Pesotum sp., P. gigantea, Tympanis spp., Trichoderma spp. and an unknown basidiomycete.     

Transfer of the P-type of Heterobasidion annosum from old-growth stumps of Picea abies to Picea abies and Larix×eurolepis

The infection of Picea abies and Larix x eurolepis by Heterobasidion annosum was studied in felled trees in Sweden. Thinnings were carried out in two stands of L. x eurolepis, 15 and 20 years old, and in a 25-year-old stand of P. abies, established on a site heavily infected by H. annosum. The 15- and 20-year-old L. x eurolepis stands exhibited the greatest incidence of butt rot with infections amounting to 38 and 57%, respectively, of removed trees. The incidence of butt rot in the P. abies stand was only 5%. Heterobasidion annosum was the main butt rot causing fungus. All isolates of H. annosum were of the P-intersterility group. Transfer of H. annosum from old-growth P. abies stumps to trees felled in the thinning of the next generation was demonstrated by somatic incompatibility tests. However, the majority of infections could not be connected with decay already established in the previous generation. In L. c eurolepis, rot columns were frequent, including also several genotypes in the same stem, and typically positioned in the inner sapwood. The few infections of P. abies were situated in the heartwood.     

Modelling the incidence of Heterobasidion annosum butt rots and related economic losses in alpine mixed naturally regenerated forests of northern Italy

A total of 2257 recently felled stumps were sampled from 22 mixed naturally regenerated forest stands in the Aosta Valley, western Italian Alps, and examined for Heterobasidion annosum butt rots. Disease incidence ranged from 6% to 71% depending on sites, and H. annosum accounted for 92% of the total number of diseased stumps. H. annosum incidence was significantly higher (Chi‐squared multiple comparison tests, p < 0.05) on Norway spruce (44%) than on the other tree species (silver fir = 18%, larch = 12% and Scots pine = 6%). Based on the information on the airborne inoculum composition of the fungus, all the three European species of H. annosum were present, with a variable frequency, depending on site. A partial least squares regression analysis showed that the relative abundance of Norway spruce and four variables describing the size of trees were the best predictors for the incidence of H. annosum butt rots. A model having these variables as predictors was developed (r² = 0.75; p < 0.001) and successfully validated on five additional forest stands. An estimate of the losses in yield and value of timber associated with the disease, i.e. direct losses, was also carried out in the forests included in the survey. The percentage of direct financial losses was either comparable or lower than the average level of disease incidence.     

Pathogenicity of Spanish isolates of Heterobasidion annosum s. s. in Pinus pinaster seedlings

The aim of this study was to test the pathogenicity of two Spanish isolates of Heterobasidion annosum sensu stricto in 2‐year‐old Pinus pinaster seedlings. Two types of inocula (woodchips and sawdust) were used to infect the seedlings by two different routes (stem inoculation and soil infestation). The mortality rates of the stem‐inoculated seedlings differed significantly from controls, but those of the seedlings infected via soil infestation did not differ. For both types of inoculation, the lesions were longer, and wilting symptoms were more severe in the seedlings inoculated with H. annosum than in control seedlings. For stem inoculation, biomass allocation did not differ significantly between the infected and control seedlings. However, the percentage of fine roots was lower in seedlings infected via soil infestation than in the control seedlings. To our knowledge, this is the first pathogenicity test with H. annosum isolates and P. pinaster.     

The effect of nitrogen fertilization on fungistatic phenolic compounds in roots of beech (Fagus sylvatica) and Norway spruce (Picea abies)

The effect of nitrogen fertilization on fungistatic phenolic compounds in fine roots of beech and Norway spruce growing in afforestation plots was analysed. The plots were situated at two sites in Switzerland on acidic soil with low base saturation. For 9 years, the trees have been treated with dry ammonium nitrate to give 0, 10, 20, 40, 80, 160 kg N ha^?1 year^?1, respectively. The phenolic compounds responded differently to fertilization. Fine roots of beech showed a significant decrease of (?)‐epicatechin and piceatannol with increasing nitrogen fertilization. The concentration of protocatechuic acid was increased with fertilization. Roots of fertilized Norway spruce showed significantly decreased concentrations of 4‐hydroxyacetophenone and piceatannol. The mycelial growth of three isolates each of Heterobasidion annosum s.l. and Cylindrocarpon destructans was tested on agar media containing various phenolic compounds in concentrations found in fine roots of Norway spruce (Picea abies) and beech (Fagus sylvatica). All three H. annosum isolates were inhibited by p‐coumaric acid and (?)‐epicatechin. Two isolates were inhibited by another four phenolic compounds (p‐hydroxybenzoic acid, 4‐hydroxyacetophenone, piceatannol and protocatechuic acid), one by (+)‐catechin. Two of three C. destructans isolates were inhibited by all phenolic compounds except for (+)‐catechin which affected only one isolate, one isolate did not respond at all.     

Virulence and double‐stranded RNA in Sphaeropsis sapinea

Forty wildtype isolates of Sphaeropsis sapinea were grouped into the morphotypes A and B based on previously defined differences in cultural and morphological criteria as well as restriction sites for Dde I and Bst UI endonucleases in nuclear ribosomal DNA amplicons. Thirteen of 20 type A isolates and nine of 20 type B isolates contained detectable dsRNA (55%) of different molecular weight and size. dsRNA was transmitted into conidia at a frequency of 71–100%. By selecting single conidia, dsRNA‐free subcultures were obtained from six of 22 isolates containing dsRNA. Pathogenicity tests on expanding buds of landscape trees of three species of Pinus showed highly significant statistical interactions between isolate virulence, Pinus species, and year. Pine species‐year had a profound impact on virulence. The pattern in the interactions was revealed by principal component analysis of the interaction sums of squares of the anova (Additive Main Effects and Multiplicative Interaction; AMMI). Pinus sylvestris was highly interactive in its susceptibility to S. sapinea with seasonal effects. P. nigra and P. resinosa were more stable. The interactivity analysis was used to apportion interaction to specific isolates to improve the accuracy of the estimates of virulence. Estimates of the relative virulence of isolates were predicted over five different Pinus species‐years. Isolates were ranked in virulence and interactivity using the AMMI model. This model permitted mean separation tests of the relative virulence among isolates over the combined Pinus species‐years. One isolate was identified as potentially having dsRNA‐mediated hypovirulence based on the significantly greater virulence of its isogenic, dsRNA‐free subculture, as expressed over the three Pinus species and 2 years. Type A isolates containing dsRNA ranged from stable to highly interactive and from low to high in virulence. Type B isolates containing dsRNA were similar in interactivity but virulence ranged from avirulent to moderate, seldom exceeding the mean for S. sapinea. dsRNA‐free isogenic subcultures tended not to express higher virulence than their dsRNA‐containing parent strains but often changed in interactivity. Therefore, in one year a dsRNA‐free subculture might be more virulent than its dsRNA‐containing parent. In another year the dsRNA‐free subculture might be less virulent.     

Persistence of a biocontrol strain of Phlebiopsis gigantea in conifer stumps and its effects on within‐species genetic diversity

Fungal isolations and genetic fingerprinting were used to determine whether Phlebiopsis gigantea stump treatment against Heterobasidion annosum sl. using a single genotype (Rotstop) would affect the genetic diversity of P. gigantea populations. The survival time of P. gigantea was longer in Norway spruce (Picea abies) stumps compared to Scots pine (Pinus sylvestris) as no isolates were obtained from pine stumps 6 years after treatment, whereas in about half of the spruce stumps the fungus was still present. The usage of Rotstop did not seem to increase the occurrence of the fungus 5 years after the treatment in fresh (1‐year‐old) untreated stumps within the same forest stands. All the isolates from the 6‐year‐old treated spruce stumps were identical in genotype with the Rotstop‐strain, whereas all isolates from the fresh untreated spruce and pine stumps differed from it. Within the treated pine stand, the biocontrol usage seemed to have caused a slight reduction in genetic markers not related to Rotstop, but there were no statistically significant differences between the marker frequencies and the local natural population. Thus, Rotstop is not likely to cause any immediate threat to the genetic diversity of P. gigantea.     

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.     

Persistence of Cryphonectria hypoviruses after their release for biological control of chestnut blight in West Virginia forests

Although Cryphonectria hypoviruses have been relatively successful as biological control agents of chestnut blight in Europe, their success in North America has been limited. Experimental releases of hypoviruses were made in 1978–82 at two sites in West Virginia forests with high densities of regenerating chestnut trees. Cryphonectria hypovirus 1 (CHV‐1) from Europe, as well as American isolates of Cryphonectria parasitica containing CHV‐3, were used for these releases. Although most trees died during the 5‐year release period, it was not known if the hypoviruses persisted in the C. parasitica population at the two sites. When the experimental plots were revisited in 1994, few chestnut trees were found. The exception was one plot containing coppice sprouts that had grown from the root collars of the original trees. The authors intensively sampled C. parasitica from experimental plots and screened recovered isolates for double‐stranded RNA (dsRNA). None of the isolates contained CHV‐1; only six isolates contained CHV‐3, all from the plots with the coppice sprouts. CHV‐4, which occurs naturally in West Virginia forests and in two released isolates, hybridized to dsRNA from the isolates containing CHV‐3, indicating mixed infections. CHV‐4 also hybridized to dsRNA from other isolates sampled inside and outside the treated plots. In contrast to CHV‐1 and CHV‐3, however, CHV‐4 has little effect on the growth or phenotype of C. parasitica. The limited persistence of CHV‐1 and CHV‐3 may have resulted when the C. parasitica population was reduced in size due to the failure of chestnut trees to resprout because of competition from other hardwood species.     

Effect of inoculum density and borate concentration in a stump treatment trial against Heterobasidion annosum

The effectiveness of disodium octaborate tetrahydrate (DOT) as a stump treatment chemical for Sitka spruce in Britain was tested on six occasions by inoculating treated and untreated stumps with basidiospores of Heterobasidion annosum at three concentrations ranging from an average 49 viable spores/ml to 4.9 × 10⁵/ml of water. The extent of colonization of heartwood by H. annosum was measured and, along with the incidence of infected stumps, provided a measure of the combined effects of spore concentration and of the two DOT treatments (15 and 30 g/m²) on the trial results. On untreated stumps, both the incidence of infection and the cross‐sectional area of stump heartwood colonized by the fungus increased with inoculum density. The same effect was evident in treated stumps, but it was reduced by increasing DOT application. Infection was at its lowest in stumps treated with DOT at 30 g/m², being entirely absent from those 60 stumps that were inoculated with the fewest spores. The implications of these findings for the design of trials of control agents that rely on artificial inoculation with H. annosum and for the selection of dose rates to use in harvesting operations are discussed.     

Viruses of Heterobasidion parviporum persist within their fungal host during passage through the alimentary tract of Hylobius abietis

Heterobasidion annosum (Fr.) Bref. sensu lato is an important fungal parasite of coniferous trees throughout the temperate regions of the world. Approximately 15% of Heterobasidion isolates are infected by dsRNA viruses, which are considered as obligately intracellular and transmit vertically into both basidiospores and asexual conidia. Insects such as H. abietis and its larvae feeding on wood colonized by Heterobasidion fungi may carry Heterobasidion conidia and hyphae. In this study, we used H. abietis as a model species to reveal whether Heterobasidion viruses resist in their fungal host passing through the digestive tract of insects that may potentially serve as disseminators of fungal propagules. Pinus sylvestris branches were inoculated by three different strains of Heterobasidion parviporum, hosting taxonomically diverse virus species: Heterobasidion RNA virus 2, HetRV4 and HetRV6. Then, the inoculated branches were fed to H. abietis insects, and beetle excrements were investigated for Heterobasidion infections. All the inoculated fungal strains survived passage through the alimentary tract of the insects (survival rate 25–80%). The passage rates of the viruses inside their hosts varied considerably, ranging from 0 to 67%. Two different mycoviruses, HetRV2 and HetRV6, survived the intestinal passage of their fungal host, while the virus species HetRV4 was detected among none of the five H. parviporum isolates retrieved from insect faeces. The relative stability of fungi harbouring viruses suggests that if viruses are to be used for biological control against Heterobasidion species, it is likely that insect feeding does not considerably decrease the virus effect, but might instead enhance short‐range dispersal of the viral biocontrol agent.