Experiments on the susceptibility of conifers to Heterobasidion annosum in Great Britain

During the period 1960–71, experimental plantings were established at three sites in western Britain that were infested with Heterobasidion annosum: Ceri in mid‐Wales, Lael in north‐west Scotland and Red Marley in the West Midlands of England. At each site a randomized block experiment involving at least four species was supplemented with an ancillary trial of other species. In two of the experiments various treatments were applied to the previous stand of trees before or at felling, but only stump removal reduced the amount of disease in the succeeding crops. At Ceri, the incidence of H. annosum in stems removed at first thinning was: Picea sitchensis, 14%; Pseudotsuga menziesii 11%; Pinus contorta 3% and Abies procera 1%. At Lael, the figures were Larix decidua 59%; P. menziesii 51%; Chamaecyparis lawsoniana 37%; Abies amabilis 33% and Tsuga heterophylla 21%. There was negligible disease in A. procera;Abies grandis and Pinus sylvestris. At Red Marley, the incidence of disease was: P. menziesii 28%; T. heterophylla 18%; A. grandis 7%; Picea abies 1% and Pinus nigra var. maritima 0%. In the ancillary trial at Lael, the incidence of H. annosum in P. sitchensis was 55% and in P. abies 16%. The mean height of colonization by H. annosum within the diseased stems removed at first thinning at Lael (age 21–22 years) was 2.1 m for L. decidua, 1.4 m for P. sitchensis and 1.3 m for P. abies. Armillaria sp. caused mortality and decay in two of the experiments and these data are also presented. The results are discussed in relation to other information on the susceptibility of these species to H. annosum in the UK and elsewhere.     

Infection studies with Heterobasidion annosum on young trees of Picea abies

Infection studies with Heterobasidion annosum were carried out on 15-year-old, cloned spruces between 1971 and 1978. The inoculum was introduced into the sapwood of the test plants at the base of the stem. All inoculations, conducted at different times of the year, were uniformly successful. The pathogen spread through the xylem tissue rapidly during the vegetation period, but from early autumn onwards gradually disappeared over the following two years. After this period, the fungus could no longer be detected.     

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.     

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.     

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.     

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.     

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.     

Heterobasidion annosum: Development of Butt Rot following Thinning in two young First Rotation Stands of Norway Spruce

Severe butt rot caused by Heterobasidion annosum (Fomes annosus)developed in two stands of first rotation Norway spruce on oldagricultural land within 11 years of first thinning. At SiteI (Devon), 11 per cent of thinning stumps and 23 per cent ofclear felled stumps were found to be decayed when examined soonafter the crop was felled at age 37 years. It is estimated thatbetween 4.4 per cent and 6.6 per cent of potential volume waslost on clear felling. Significant butt rot had also developedin a 33-year-old stand of Norway spruce in Site II (Aberdeenshire)11 years after a Scots pine nurse crop was removed. Here, thedisease was found to be widespread on an area of 2 ha, and 73per cent of trees had butt rot. Decay extended 3 m up the stems,and occupied significant areas of the butt. In both cases, thepresence of butt rot in the crops led to premature clear felling,which resulted in additional loss of potential revenue. It seems most likely that the disease became established followinginfection of thinning stumps by airborne spores of the fungus:at Site I, the thinning stumps were not protected whilst atSite II it would appear that a chemical or biological treatmentapplied to the pine stumps failed. The subsequent rapid developmentof the disease was due, in part, to a variety of environmentaland edaphic factors.     

Responses of Sitka spruce of different genetic origins to inoculation with Heterobasidion annosum: lesion lengths,fungal growth and development of the lignosuberized boundary zone

Three genetically distinct groups of Sitka spruce, open‐pollinated Queen Charlotte Island provenance, A13 selected seed and M0044 half‐sib mixture, were wounded alone or wounded and inoculated with Heterobasidion annosum sensu stricto on the lower stems. Growth of the pathogen and lesion formation was compared in the three genetic groups after treatment. No differences in the rate of colonization of the three genetic groups were observed over a 40 day period; lesion lengths in the bark and sapwood correlated closely. Moreover, lesions were considerably longer in inoculated plants than in those which were wounded only. No correlations were found within or between host genetic groups in the numbers or total areas of resin canals present in the first 18 mm from the wound in bark tissues for the three host genetic groups. Formation of the ligno‐suberized boundary zone (LSZ), however, was inhibited in the bark of inoculated plants, being first detected later and at a greater distance from the wound/inoculation point in the presence of H. annosum than in plants that were wounded only. Thickness of the suberin cell layers within the LSZ of M0044 plants was greater in wounded and inoculated, than in wounded only plants. The significance of these findings in relation to detecting spruce genotypes potentially resistant to H. annosum is discussed.     

Pathogenicity of Heterobasidion annosum (Fr.) Bref. sensu stricto on coniferous tree species in Turkey

Two‐year‐old seedlings of Pinus brutia, P. brutia var. eldarica, Pinus pinea and 3‐year‐old seedlings of Pinus radiata, Pinus sylvestris, Pinus nigra and Cedrus libani were inoculated on the lower stem with isolates of Heterobasidion annosum s.s. collected from the Black Sea and Mediterranean regions of Turkey. In total, 315 seedlings were inoculated in April 2014 and incubated in a growth chamber for 7 weeks at 18–20°C. All isolates were pathogenic on the seven different hosts and had the ability to grow in living sapwood. The isolates had a greater growth on C. libani, P. sylvestris and P. radiata seedlings compared to plants of the other species tested. The least affected species were P. brutia and P. nigra. The isolates originating from the Black Sea region caused longer lesions on the hosts. Overall mortality during 7 weeks of incubation was 4%.     

Growth inhibitors produced by the in vitro culture of Heterobasidion annosum

The optimum conditions of in vitro production of growth inhibitory substances by Heterobasidion annosum are determined. Some terms and derivation of some functions are described in connection with the phases of in vitro production of growth inhibitory substances.     

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.     

Spatial distribution and persistence of Heterobasidion parviporum genets on a Norway spruce site

Spatial distribution of Heterobasidion genets over a period of ca 50 years in two successive generations of Norway spruce (Picea abies) was unravelled. The genets were first identified in 1993 in a naturally regenerated 43‐year‐old spruce stand that had been thinned the previous winter. Heterobasidion parviporum was found in 17.5% of the old stumps of the previous spruce generation. Nine genets were identified on the study plot; seven of them were present in old stumps of the previous tree generation and two only in the new spruce generation. Eighteen spruce trees of the new generation were infected, 15 of them by vegetative growth of genets originating from the old stumps. The study plot was investigated again in 2005. No new genets had been established after thinning, and three old genets had died out. The remaining genets had infected five new trees, most likely from the thinning stumps of diseased trees. At the age of 56 years, 16.1% of the residual spruces were infected by Heterobasidion. The results of this study suggest that if spore infection to stumps of spruce can be prevented, the decay frequency caused by H. parviporum will not necessarily increase in successive generations.     

Estimation of dispersal gradients of S- and P-type basidiospores of Heterobasidion annosum

The number of airborne deposits of the root pathogen Heterobasidion annosum was counted with a wood disc method at distances 5–1000 m from one S-type and one P-type infection centre in stands of Picea abies in June, July and August, 1995. The level of background deposition in the area was determined in four healthy control stands. Based on intersterility group determination of deposits, only measurements within 100 m of the infection centres could be used in estimations of dispersal gradients owing to confusion with background deposition. A power law gradient model adequately described the reduction of deposits with increasing distance from the infection centres. Gradients were fairly similar in four directions around the infection centres as the wind speeds were low (3.2m/s) during the collection periods. The gradients were steeper around the P-type centre, probably due to its lower spore production capacity. The background deposition level (2.8 colonies/m²× h) was reached at distances 1255 m and 98 m from the S-type and P-type centre, respectively.     

Compartmentalization of decay associated with Heterobasidion annosum in roots of Pinus resinosa

Decay associated with Heterobasidion annosum was compartmentalized in roots of Pinus resinosa.     

Incidence of Heterobasidion annosum in precommercial thinning stumps in coastal British Columbia

Coniferous stumps in 83 stands in coastal British Columbia were sampled 3-5 years after precommercial thinning. The percentage of stumps and surface area colonized by Heterobasidion annosum were determined for 25 stumps of each species in each 5-cm diameter class present in each stand. There were significant differences among species in the percentages of stumps and surface area colonized, with Douglas-fir (Pseudotsuga menziesii) having the lowest values, amabilis fir (Abies amabilis) and Sitka spruce (Picea sitchensis) the highest and western hemlock (Tsuga heterophylla) being intermediate. For stumps of each species 5–20 cm in diameter, both the percentage of stumps and surface area colonized increased with increasing diameter. In stumps that were grafted to an adjacent tree, there was decreased incidence of H. annosum for Douglas-fir and Sitka spruce and increased incidence for western hemlock and amabilis fir. There were trends in the percentage of stumps and area colonized for season of thinning and biogeoclimatic subzones with the values for most species decreasing as the amount of precipitation increased. Colonization of precommercial thinning stumps by H. annosum occurs throughout the coastal region of British Columbia, and this will increase the amount of inoculum and will likely increase the incidence of butt rot. The results of this study suggest that the increase in inoculum can be minimized by thinning before age 15, by cutting only trees less than 10 cm in diameter and by thinning during low risk seasons.     

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.     

Variation of RAMS markers within the intersterility groups of Heterobasidion annosum in Europe

Random amplified microsatellite (RAMS) markers were used for the analysis of genetic variation within and among the intersterility groups of Heterobasidion annosum. Isolates from seven European countries were used, and the analysis of 77 markers revealed banding patterns with several markers unique to intersterility (IS) groups S, P and F. The highest number of polymorphic markers and the lowest number of fixed markers was observed within IS group P. In addition, a low level of geographic differentiation was observed within IS groups S and P by amova analysis.     

Root and butt rot caused by Heterobasidion annosum in Atlantic coniferous ecosystems of Spain

In the second half of the 20th century, the forested surface in northern Spain started to increase during a process of reforestation and the replacement of native forest. This reforestation was performed mostly by introducing monocultures of exotic coniferous species. One of the implications of intense forest exploitation and the introduction of new forest species is an increase in disease outbreaks. Because the genus Heterobasidion includes some of the most significant conifer pathogens in the world, surveys were conducted to collect and identify H eterobasidion isolates associated with diverse hosts within coniferous forests and plantations in the Basque Country, northern Spain. A total of 159 stands were surveyed, and 45 isolates were obtained from different trees. Based on sequencing of the internal transcribed spacer region of the ribosomal DNA, all collected isolates were identified as European H eterobasidion annosum s.s. (European P‐type). Heterobasidion annosum was detected in 28.3% of the sampled stands, with the following distribution by host: Chamaecyparis lawsoniana (11.1%), P inus pinaster (2.2%), P . radiata (42.2%), P . nigra (2.2%), P . sylvestris (17.8%), P seudotsuga menziesii (17.8%) and P icea abies (6.7%). The spatial distribution of the population showed a high degree of clustering. This is the first report of H . annosum s.s. causing damage to forest plantations of Chamaecyparis lawsoniana, P inus pinaster, P . radiata, P seudotsuga menziesii and P icea abies in Spain. In the current context of forest pathosystems, the management practice of replacing susceptible forest species with resistant species is recommended.