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.     

Heterobasidion abietinum on Abies species in western Turkey

Forty‐five basidiocarp specimens of Heterobasidion were collected from native Abies species in three locations in western Turkey: A. nordmanniana ssp. bornmülleriana in Bolu province, A. nordmanniana ssp. equi‐trojani in Balikesir province and A. cilicica in Antalya province. Pure cultures were isolated from the basidiocarps and identified to the species level with the aid of mating tests. All the specimens proved to belong to the species Heterobasidion abietinum. This root rot fungus is common in the forests investigated and appears to be relatively virulent on Abies in Turkey. This is the first report of H. abietinum outside Europe.     

Gene flow and inter‐sterility between allopatric and sympatric populations of Heterobasidion abietinum and H. parviporum in Europe

The tree pathogenic fungi Heterobasidion parviporum and H. abietinum are recently described sibling species that show marked host preference for Picea abies and Abies alba, respectively. H. parviporum shows a higher reproductive character displacement (i.e. higher inter‐sterility) towards H. abietinum in sympatry than in allopatry. We inspected whether this pattern was also present in H. abietinum by studying the inter‐sterility and gene flow amongst sympatric central European populations of H. abietinum and H. parviporum, and two isolated allopatric populations, Pyrenean H. abietinum and Scandinavian H. parviporum. Inter‐ and intra‐specific fixation index values were calculated from DNA sequence data of the GST‐1 locus and four microsatellite loci. Present allopatric and sympatric populations of H. abietinum were equally inter‐sterile towards H. parviporum, not suggestive of relaxed reinforcement. Several hypotheses explaining the observed pattern are presented. Genetic differentiation was observed between H. abietinum in the Pyrenees and in the Alps, suggesting geographical structure of H. abietinum in Europe.     

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.     

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.     

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.     

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.     

Genotypic diversity and distribution of Sphaeropsis sapinea within Pinus radiata trees from northern Spain

Sphaeropsis sapinea is an important latent pathogen of Pinus spp., outbreaks of which have a considerable impact on plantations. This study considers the population diversity and distribution of S. sapinea in northern Spain at different spatial scales from single plantations to a wide area covered by Pinus radiata trees. Estimation of genotypic diversity is an important component of the analysis of the genetic structure of plant pathogen populations. Ten simple sequence repeat (SSR) markers were used, together with vegetative compatibility tests, to study the genetic diversity among S. sapinea isolates. Polymorphism analysis at SSR loci is a simple and direct approach for estimating the genetic diversity of S. sapinea isolates. From a total of 86 isolates collected from four different areas, 14 microsatellite haplotypes and 13 vegetative compatibility groups (VCGs) were identified. The percentage of maximum genotypic diversity, based on Stoddart and Taylor's index, for microsatellites of the northern Spain population ranged from 14.6% to 38.1% and from 8.0% to 29.4% for VCGs. Analysis of these markers and vegetative compatibility groups confirmed that S. sapinea reproduces mainly asexually due to its reduced genotypic diversity in spatially close populations. Isolates of S. sapinea from northern Spain populations were predominantly monomorphic at the tested SSR loci. Vegetative compatibility groups also indicate a low level of genetic variability in these samples, which appear to be clonal.     

Local population structure of Hymenoscyphus fraxineus surveyed by an enlarged set of microsatellite markers

The population structure of Hymenoscyphus fraxineus, the causal agent of ash dieback, was assessed at four closely located sites in the Czech Republic. To analyse the genetic variation, one Swiss and one Norwegian population with known population structures were selected as reference points. The analysis was performed using 16 previously published and five newly developed microsatellite markers. The quality of the new markers was assessed by sequencing the flanking region, identifying the type of the mutation and analysing the inheritance and linkage between all pairs of loci. In addition, markers were tested on 45 Japanese strains to confirm their usability on native H. fraxineus populations in Asia. One of the new markers was monomorphic in all European populations and one marker exhibited a high percentage of null alleles in the Japanese samples. Twenty markers in the four Czech populations showed lower average gene diversity than in the other two European populations. One Czech population significantly differed from all the others, with a pairwise G_ST of approximately 0.2, the lowest allelic richness and very low average gene diversity, likely resulting from a founder effect. The amount of genetic differentiation between the four neighbouring Czech populations exceeds the expected value from previous Europe‐wide population studies and suggests that local population dynamics can affect the population structure of H. fraxineus.     

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.     

Heterobasidion annosum group S identified in north-eastern China

Samples of Heterobasidion annosum were collected in two stands of mixed forest in Changbaishan Nature Reserve in north-eastern China. Pure cultures were isolated from 15 specimens growing on Abies, Picea, Pinus and Populus. Mating tests showed that all these collections belong to the S inter-sterility group. The compatibility of Chinese homokaryons with S homokaryons from Europe was 100%. Clamp connections appeared almost equally in both strains of each pairing, and no distinct demarcation line formed in the pairings. The mating frequency of Chinese isolates with the European F group was also high, approximately 94%. These matings, however, were usually unilateral and a more or less distinct demarcation line was present in most of the pairings. The Chinese isolates were completely intersterile with the European P group. The S group of H. annosum in north-eastern China appears to have similar morphology and ecology as in Europe. It is common on Abies and Picea, but is not a serious pathogen in the pristine forests of north-eastern China.     

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.     

Single sequence repeat markers reflect diversity and geographic barriers in Eurasian populations of the conifer pathogen Ceratocystis polonica

The blue‐stain fungus and vascular stain pathogen Ceratocystis polonica and its associated bark beetle vectors, particularly Ips typographus and I. typographus japonicus, cause significant losses to several spruce species in Eurasia. Nothing is, however, known about the population genetics of this conifer pathogen. In this study, a set of single sequence repeat (SSR) markers were developed to determine the population structure and genetic diversity of C. polonica in Europe and Japan. ISSR‐PCR primers were used to target SSR‐rich regions and specific primers were designed flanking the SSR regions found in these amplicons. The SSR primers developed for C. polonica were found to be transferable to six other Ceratocystis species from conifers, residing in the Ceratocystis coerulescens complex. Ninety‐eight isolates representing four populations of C. polonica (Austria, Norway, Poland and Japan) were tested using 10 selected polymorphic SSR markers. A high level of gene diversity was found in C. polonica as a whole (H = 0.53). Analysis of G statistics showed a low degree of population structure in Europe and a high level of gene flow between populations (Gst = 0.05, Nm = 8.5). In contrast, the Japanese and the European populations of C. polonica displayed strong genetic separation, which is likely caused by geographic isolation. The low level of population structure of C. polonica in Europe and the differentiation between the European and the Japanese fungal populations mirror previous findings for I. typographus and I. typographus japonicus, the main insect vectors of this fungus. These results support the view that the fungus and the insect have closely co‐evolved together. This study also suggests that movement of C. polonica and its vectors between Europe and Asia pose a threat to forestry on both continents and this should clearly be avoided.     

Genetic variation among 11 <Emphasis Type="Italic">Abies concolor</Emphasis> populations based on allozyme analysis

In order to obtain information on the genetic structure ofAbies concolor and the genetic variation among 11 populations introduced from America to China, allozyme analysis based on starch gel electrophoresis technology was used. 24 loci of 10 allozyme systems were mensurated, and the genetic structure and genetic diversity of the 11 populations of A. concolor evaluated. The results show that the genetic variation among is significant, and the genetic variation within A. concolor populations is more important. In contrast with other conifers, the variation of A. concolor is above the average level of conifers, and higher than the same level of Abies. The percentage of polymorphic loci (P) was 62.5%, the number of alleles per locus (A) 2.08, the number of effective alleles per locus (Ae) was 1.37, the expected heterozygosity (H) 0.204, and the Shannon information index (/) 0.351 7. There is a short genetic distance (D=0.061) and a low gene flow (Nm=0.839 4) among the 11 introduced populations ofA. concolor with high genetic variation. The genetic differentiation coefficient (Gst) was 0.229 5, which is higher than that of the mean in Abies or Pinus.     

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.     

An isolated population of Heterobasidion abietinum on Abies pinsapo in Spain

Abies pinsapo, a fir endemic to a small area in southernmost Spain, is frequently attacked in natural stands by Heterobasidion root rot. To identify the Heterobasidion species causing the disease, pure cultures of the fungus were isolated from symptomatic trees in five localities in Sierra de las Nieves Natural Park and identified by pairing tests. In addition, genets of the fungus were identified in two pure stands of A. pinsapo. All the Heterobasidion specimens collected belonged to the species Heterobasidion abietinum. The largest genet found was 57 m long, and had colonized 10 trees. The large size of the main genets implied that H. abietinum had spread via root contacts from old infections generated before the establishment of the Sierra de las Nieves National Park in 1989. Exceptionally dry summers in the last 20 years may have weakened A. pinsapo and favoured spread of the disease.     

Phylogenetic and differentiation analysis of the trunk rot pathogen Fomitiporia tsugina in North America

Fomitiporia hartigii s.l. is an important trunk rot pathogen of conifers throughout the Northern Hemisphere. In North America, this pathogen primarily attacks Tsuga, but is also found on Abies, Picea and Pseudotsuga. Previous research showed that isolates of F. hartigii from North America represent a distinct phylogenetic species, known as F. tsugina. However, that conclusion is based on limited data. To better understand the phylogenetic relationships of F. tsugina in North America, a phylogenetic analysis was performed using three loci (internal transcribed spacer, nuclear large subunit and tef1) with 23 isolates originating from the northern United States, Canada and central Europe. North American isolates formed a monophyletic group with significant statistical support, confirming previous reports that F. tsugina represents a unique phylogenetic species, distinct from European F. hartigii s.s. Population subdivision between isolates of F. tsugina from eastern (Massachusetts, Michigan, New Hampshire, New York and Wisconsin) and western (British Columbia, Colorado, Oregon and Washington) North America was investigated. Tests of gene flow and genetic differentiation based on region of origin detected significant variation (F_ST = 0.761; K_ST = 0.625, p < 0.01), suggesting gene flow between the two populations may be limited. Neutrality tests revealed significant, negative departures from the standard neutral model, which could indicate that a purifying or stabilizing selection has maintained low levels of polymorphisms in the population, perhaps favouring an advantageous phenotype. Further studies are required to better understand the occurrence of F. tsugina on Abies and Picea in boreal forests outside the natural range of Tsuga.     

Tree mortality and habitat shifts in the regeneration trajectory underneath canopy of an old-growth subalpine forest

To better understand tree regeneration trajectories and the resultant coexistence of Abies with co-dominants, Picea jezoensis var. hondoensis, Tsuga diversifolia and Betula ermanii, in an old-growth subalpine forest, we investigated spatial mortality patterns during the regeneration of Abies mariesii and A. veitchii, which are abundant in the understory reflecting their shade tolerance. Regeneration of these Abies spp. from shaded understory to canopy status is affected by other canopy co-dominants. Snags of understory Abies spp. were common, suggesting that the primary mortality agent is suppression by the overstory. Although live, small Abies trees in the understory were positively associated with a Picea canopy, the long-term survival was reduced among Abies trees close to the canopy, suggesting that shading by large Picea in the overstory negatively affects understory Abies plants. The existence of shade-intolerant canopy co-dominants such as Picea and also Tsuga, which are larger and longer lived than the shade-tolerant Abies, may play an important role in preventing the Abies spp. from competitively displacing these other tree species, which are much rarer in the understory, though common in the canopy. Moreover, in spite of the fact that Betula canopies fostered recruitment and growth of Abies saplings, Abies showed no association with Betula canopy and their survival at later-stage was rather reduced near or beneath Betula canopies at the subsequent understory small tree stage. Based on spatially significant events related to tree death, this study detected such “habitat shifts” in the trajectory of tree regeneration. Accordingly, it can be concluded that careful consideration of the regeneration habitat is required for a fuller understanding of ecological processes in spatially complex old-growth forest systems.     

Genetic diversity of sea buckthorn (Hippophae rhamnoides) populations in northeastern and northwestern China as revealed by ISSR markers

As a N₂-fixing tree species, sea buckthorn (Hippophae rhamnoides) is well adapted to arid regions and is utilized for multiple purposes in China. Current knowledge of genetic variability of H. rhamnoides is limited in terms of rangewide distributions. Eleven natural populations of sea buckthorn in northeastern and northwestern China were analyzed to detect genetic variation among and within populations, by use of ISSR (inter-simple sequence repeats) markers. Using eight primers, 207 polymorphic loci were observed, ranging in size from 250 to 2500 bp. The coefficient of gene differentiation (Gst = 0.0679) showed that the total molecular variance of 11 populations was mainly existed within populations. The genetic variation within and among the 11 populations was 93.21 and 6.79%, respectively. No significant correlation between genetic and geographic distances of the populations was found using ISSR markers. Our study provides a population-level genetic profile for further investigation and conservation of genetic diversity of sea buckthorn.