Analysis of microtubule and microfilament distribution in Pinus sylvestris roots following infection by Heterobasidion species

Cytoskeletal dynamics play a crucial role in pathogen recognition and cell defence during the initial interactions between an invader and plant host. The aim of the work reported here was to characterize how Heterobasidion annosum s.s., Heterobasidion parviporum, and Heterobasidion abietinum affect the microtubules and microfilaments of Pinus sylvestris root cells 12‐, 24‐, 48‐, and 96‐h post‐inoculation. Inoculation of P. sylvestris with H. parviporum or H. abietinum, which have a lower specificity for P. sylvestris than H. annosum s.s, resulted in greater reorganization of host microtubules during the early stages of interaction than inoculation with the more specific H. annosum s.s. In some infected cells, spots of actin aggregates were observed. Disruption of cytoskeletal components by the application of specific cytoskeletal inhibitors facilitated the entry of the H. parviporum and H. abietinum into roots. These results suggest that the P. sylvestris cytoskeleton plays a role in the host response in the initial stages of the host–pathogen interaction.     

Investigations on Heterobasidion annosum s.lat. in central and eastern Asia with the aid of mating tests and DNA fingerprinting

To investigate the taxonomy of Heterobasidion in Eurasia, 49 specimens belonging to H. annosum sensu lato from Asia were identified with the aid of mating tests. Most of the specimens originated from north‐eastern and south‐western China and from the Altai region in southern Siberia, but a few isolates from Kirghizia, Japan and India were also tested. In addition to mating tests, the material from China was investigated with DNA fingerprinting. Heterobasidion annosum sensu stricto was identified only from the Altai region. Homokaryotic isolates from other specimens, except the Indian ones, were sexually compatible with H. parviporum, but they also showed a high degree of compatibility with H. abietinum and with the North American S group. The isolates from SW China (eastern Himalayas) mated with about equal frequency with the European strains of H. parviporum and H. abietinum. However, the DNA fingerprinting showed that these isolates were more closely related to H. parviporum, and hence they were tentatively included in this species. The North American S group was more distant from these Eurasian taxa. Four old isolates from India mated only weakly with the members of the H. annosum s.lat. According to the species concept presented, the distribution of H. parviporum extends from western Europe through southern Siberia to northern China, Japan and the eastern Himalayas. H. annosum s.str. is so far identified only from the Altai region outside Europe, and H. abietinum only from Europe.     

Genetic variation in Heterobasidion abietinum populations detected with the M13 minisatellite marker

Amplification profiles of the M13 minisatellite core sequence from 47 isolates of Heterobasidion annosum s.l., collected mostly from Abies alba in different European countries, were analysed in comparison with profiles of four isolates of H. annosum s.s. and three isolates of H. parviporum. Genetic variation within and among groups of populations was studied by analysis of molecular variance. A dendrogram constructed with the Neighbor‐Joining method differentiated the three species from each other. Isolates of H. abietinum from Balkan peninsula, Italian peninsula and French Pyrenees each separated into clusters according to geographical origin. Such clear geographic clustering was not detected among isolates from central Europe. The variations between H. abietinum isolates in relation to the migration history of Abies are discussed.     

Genetic variation of Heterobasidion abietinum in Southern Europe and the Mediterranean Basin

Heterobasidion abietinum causes root and butt rot on Abies species occurring in central Europe and the Mediterranean Basin in a distribution extending from Spain in the west to Turkey and Caucasia in the east. To investigate the genetic diversity within H. abietinum, 95 isolates from different regions were analysed with 61 polymorphic molecular markers. The markers showed genetic differentiation amongst the H. abietinum populations. The most diversified population is that on A. pinsapo in southern Spain, which is isolated from gene flow with other populations. Lower but still significant differences were found amongst other European populations. Isolates from Turkey showed little diversification compared with the south European isolates from east of the Alps. The genetic structure of the H. abietinum populations in relation to postglacial recolonization of Abies is discussed.     

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.     

Terpene response of Picea abies and Abies alba to infection with Heterobasidion s.l.

The monoterpene composition of Picea abies and Abies alba resin was analysed in relation to growth by Heterobasidion spp. Fifteen‐year‐old P. abies and A. alba trees were inoculated on branches with three species of Heterobasidion annosum s.l. After 4 months of incubation, each host was colonized to a significantly greater degree by the pathogen specific to that host (H. parviporum on P. abies, H. abietinum on A. alba) than by the other fungi. Analysis of the enantiomeric monoterpene profiles in the spruce and fir showed that the response in terms of the relative proportions of the monoterpene compounds in the resin differed between tree species. Following challenge with Heterobasidion spp., A. alba trees did not show changes in monoterpene composition in addition to those in the wounding response (increase in (−)‐α‐pinene and (−)‐camphene, and decrease in β‐phellandrene). In P.abies, (−)‐α‐pinene, (+)‐α‐pinene and δ‐3‐carene increased following Heterobasidion attack but not after wounding alone.     

Mycelial growth and exclusion of Heterobasidion parviporum inoculated in branches of 15‐year‐old Picea abies clones

Twigs of 22 clones of 15‐year‐old Picea abies grafts were inoculated with Heterobasidion parviporum in July and in September 2003. Fungal growth varied between clones and exclusion of the pathogen varied significantly among clones inoculated in July. The results indicate that twigs exhibit the same infection pattern as cuttings when inoculated with H. parviporum and that exclusion of the pathogen in twigs is a clone‐dependent character that occurs during the vegetation period.     

Cedrus libani: the most susceptible Turkish conifer species to local Heterobasidion isolates in spring inoculations

Five‐year‐old seedlings of Pinus nigra, Pinus brutia, Abies nordmanniana ssp. bornmülleriana and Cedrus libani, 3‐year‐old seedlings of Pinus sylvestris, and 2‐year‐old seedlings of Juniperus excelsa were inoculated on the lower stem with Turkish Heterobasidion abietinum and Heterobasidion annosum s.s. isolates. In total, 300 seedlings were inoculated in April 2007 with five isolates of each Heterobasidion species and incubated in a glasshouse for 12 weeks. The daily maximum temperature increased gradually from 13°C to 31°C by the end of the incubation period. Infection incidence, mortality, lesion length in the inner bark and fungal growth in the xylem were examined. For H. abietinum, infection incidence ranged from 0% to 36%, and for H. annosum s.s. from 0% to 60%. Mortality was low; only 1% of the inoculated seedlings died. Mean lesion lengths varied between 7.6 and 11.9 mm for H. abietinum and 8.0 and 15.7 for H. annosum s.s. in all species apart from C. libani, in which the corresponding values were 42.2 and 42.6 mm respectively. Similarly, mean fungal growth in sapwood of C. libani was approximately 25 mm for both Heterobasidion species, in contrast to 0–3.2 mm in the other tree species. The H. abietinum isolates were not reisolated from P. nigra, P. sylvestris or J. excelsa. The H. annosum s.s. isolates did not infect Abies seedlings. Only P. brutia and C. libani were susceptible to both pathogens. Control seedlings showed no symptoms. The results indicated that C. libani is highly susceptible to Turkish isolates of Heterobasidion.     

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

In 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.     

Growth rates of Heterobasidion annosum s.s. and H. parviporum in functional sapwood of Pinus sylvestris and Picea abies

Growth rates of H. annosum s.s. and H. parviporum were investigated in the functional sapwood of young Pinus sylvestris and Picea abies plants as an indicator of the relative susceptibilities of the hosts to these pathogens. The stems of 520 five‐year‐old P. abies and 321 four‐year‐old P. sylvestris plants were inoculated and the extent of infection determined 16 weeks later. H. annosum s.s. grew further than H. parviporum in P. sylvestris sapwood, while in P. abies, no differences between the two Heterobasidion spp. were found. Both H. annosum s.s. and H. parviporum spread faster in the sapwood of P. abies than in P. sylvestris. There was high within‐host species variation in growth rates for both P. sylvestris and P. abies suggesting it may be possible to identify tree genotypes with lower susceptibility.     

Heterobasidion on Abies nordmanniana in north‐eastern Turkey

Occurrence of Heterobasidion annosum s.l. was investigated in 15 coniferous stands in the Giresun and Pontic Mountains in north‐eastern Turkey. Basidiocarps of the fungus were found in 11 stands. Fifty‐two basidiocarp specimens of Heterobasidion were collected from stumps of Abies nordmanniana ssp. nordmanniana and two from stumps of Picea orientalis. Pure cultures were isolated from the basidiocarps and identified with the aid of mating tests. Forty‐five (90%) of the specimens collected from A. nordmanniana were identified as H. abietinum and five (10%) as H. annosum s.s. The former species was also found twice on P. orientalis. This is the first report of H. annosum s.s. for Turkey and the first report of H. abietinum on P. orientalis. Heterobasidion abietinum seems to be mostly a saprotroph on A. nordmanniana.     

Effect of Thinning and Phlebiopsis gigantea Stump Treatment on the Growth of Heterobasidion parviporum Inoculated in Picea abies

The spread of Heterobasidion parviporum Niemelä & Korhonen in roots of Norway spruce was studied in three unthinned first rotation stands of Norway spruce [Picea abies (L.) Karst.] on former agricultural land in south-western Sweden. Heterobasidion parviporum was inoculated at stump height into the trunk of 135 standing trees in a randomized block design. One year after inoculation, two-thirds of the trees were thinned out and one-third was left standing. Half of the stumps left by thinning were treated with spores of Phlebiopsis gigantea (Fr.) Jül and half were left untreated. The spread of H. parviporum was examined both 3 and 5 yrs after inoculation. The rate of spread of H. parviporum and the proportion of infected roots were found to be significantly higher in the root systems of the stumps than in those of the standing trees. Treatment with P. gigantea had no significant effect on the development of H. parviporum in the stumps. There was a tendency 5 yrs after inoculation, however, for a lower proportion of H. parviporum-infected roots in the stumps treated with P. gigantea than in the untreated stumps. In conclusion, thinning of infected Norway spruce was found to increase the rate of spread of H. parviporum in the root systems of the infected trees, which could increase the risk of a rapid build-up of infection in the remaining stand.     

Comparison of colonization capacity by asexual spores of Heterobasidion species in Norway spruce wood

Two species of the tree pathogenic fungus Heterobasidion spp. exist in Sweden, Heterobasidion annosum s.s. and Heterobasidion parviporum. Both species are known to infect Norway spruce (Picea abies). The aim of the study was to examine the interspecific competition between H. annosum s.s. and H. parviporum as well as their colonization rate in fresh Norway spruce wood. Equal amount of conidiospores from each species was sprayed together on 30 fresh, previously uninfected, Norway spruce billets. After incubation in a greenhouse, the proportion of Heterobasidion spp. colonies belonging to each species was recorded. Of the 196 colonies isolated from the upper part of billets, 195 were H. parviporum. All isolated colonies further down in the billets were H. parviporum. To study the colonization rate, H. annosum s.s. and H. parviporum were sprayed alone on 30 spruce billets each, incubated and growth recorded both vertically and horizontally. H. parviporum grew further down in the billets (p = 0.008) and covered a larger area (p < 0.001) than H. annosum s.s. While H. annosum s.s. and H. parviporum both infected fresh Norway spruce wood H. parviporum outgrew and outcompeted H. annosum s.s during the early colonization stage.     

Turkish Heterobasidion abietinum is pathogenic to inoculated Abies nordmanniana ssp. nordmanniana and ssp. bornmülleriana

Four‐year‐old seedlings of Abies nordmanniana ssp. bornmülleriana and 5‐year‐old seedlings of Abies nordmanniana ssp. nordmanniana were inoculated on the lower stem with 28 Heterobasidion abietinum isolates originating from four different regions of Turkey. Replicate seedlings were incubated in greenhouse and growth chamber. After 7 weeks, infection incidence, mortality, lesion length in the inner bark and fungal growth in the sapwood were examined. Infection incidence in different host–incubation combinations ranged between 70.5 and 79.5% and the average mortality rate was 4.2%. Average lesion lengths varied between 8.4 and 33.9 mm, and average fungal growth between 18.7 and 34.8 mm. There was a significant positive correlation between lesion length in the inner bark and fungal growth in sapwood on both hosts and conditions. Clear differences in virulence between H. abietinum isolates originating from different regions were not found. The results indicate that H. abietinum is pathogenic on both investigated subspecies of A. nordmanniana. Both lesion length and fungal growth were greater on ssp. bornmülleriana, indicating that it would be more susceptible than ssp. nordmanniana.     

Susceptibility of Abies pinsapo and its tree cohort species to Heterobasidion abietinum

Abies pinsapo, a rare fir found in the southernmost mountain region of Spain, is severely affected by Heterobasidion abietinum. It was hypothesized that spread of the disease might be favoured by woody species growing in the same habitat. In order to evaluate this hypothesis, artificial inoculation tests were performed on other potential hosts forming the tree cohort species with A. pinsapo. Inoculation test on seedlings demonstrated that H. abietinum was able to colonize stems of Pinus halepensis, P. pinaster and Quercus ilex subsp. rotundifolia, but failed on Q. faginea. Both Pinus species were colonized to a similar extent as the natural host, but Q. ilex subsp. rotundifolia showed a significantly lower susceptibility than conifers. These results suggest that Pinus spp. growing in mixed stands with A. pinsapo might favour spread of H. abietinum. As a consequence, natural replacement of pure A. pinsapo stands by mixed forest Abies‐Quercus should be facilitated.     

Predicting the activity of Heterobasidion parviporum on Norway spruce in warming climate from its respiration rate at different temperatures

We studied the effect of climate warming on Heterobasidion root rot in boreal forests by measuring respiration activity of pure cultures of Heterobasidion parviporum in Norway spruce (Picea abies) sawdust and by linking these data to temperature data obtained from three spruce forests located along a north‐south transect stretching from northern Germany to northern Finland. The pure cultures applied in this investigation were homokaryotic, but in a separate investigation, we found no significant difference between the activity of homo‐ and heterokaryotic isolates. We also found that the temperature response curves of growth and respiration rates of this fungus were similar and propose that respiration reflects the general activity of H. parviporum. The respiration data were scaled up to annual cumulative respiration activity using daily temperature measurements from soil and air in the spruce forest sites. The annual respiration activity of H. parviporum showed a linear relationship with the average annual air temperature. An increase in the annual air temperature by 5°C would raise the annual activity of H. parviporum in spruce roots in northern Finland, southern Finland and northern Germany by 91%, 53% and 40%, respectively. This increase remains below the predicted increase in forest growth in northern Finland but exceeds considerably the predictions for southern Finland. According to the previous literature, a number of other decay fungi show a similar activity response to temperature as H. parviporum, suggesting that this result can be generalized to decay fungi with similar ecological habits.     

Tree‐ring isotope analysis of Norway spruce suffering from long‐term infection by the pathogenic white‐rot fungus Heterobasidion parviporum

We report for the first time a tree‐ring isotopic analysis on host trees infected with Heterobasidion parviporum. By measuring carbon and oxygen stable isotope ratios in tree rings over ca. 150 years of forest growth, we obtained evidence that stomatal conductance increases in Picea abies affected by H. parviporum. We put forward this approach as a novel way of providing an insight into plant–pathogen relationships during tree life.