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.     

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.     

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.     

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.     

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.     

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.     

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.     

The effect of Phlebiopsis gigantea and urea stump treatment against spore infection of Heterobasidion spp. on hybrid larch (Larix × eurolepis) in southern Sweden

The efficacy of stump treatment with 40% urea solution and spore suspension of Phlebiopsis gigantea against primary infection by Heterobasidion spp. on hybrid larch (Larix × eurolepis) was tested in two field studies in southern Sweden. In the first study, stumps treated with urea or P. gigantea were sampled by cutting a cross‐section disc after 3 months following exposure to natural conditions, and in the second study, stumps treated with only P. gigantea were sampled after 2 months. Spore traps made from hybrid larch, Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) were used to estimate the abundance of ambient spores of Heterobasidion spp. in the second study. Urea significantly reduced infection frequency of Heterobasidion spp. compared to the control. Phlebiopsis gigantea was less effective at reducing infection frequency in the first study than in the second study. Infections were caused by both H. parviporum and H. annosum sensu stricto. The amount of H. annosum infection in proportion to Heterobasidion spp. was significantly lower on the stumps treated with P. gigantea compared to the control; however, the proportion of H. parviporum on the treated stumps was not higher than the control in the first study. Spore traps made of Scots pine had a significantly higher frequency of infection than Norway spruce and hybrid larch. Only the spore traps made of hybrid larch showed significant correlation with the control stumps in terms of relative infected area. Conclusively, it seems prudent to protect hybrid larch stumps from primary infection by Heterobasidion spp., and both urea and P. gigantea can be recommended as stump treatment agents on hybrid larch, even if urea seemed to present more stable results.     

Respiration rate determinations suggest Heterobasidion parviporum subpopulations have potential to adapt to global warming

Trees are known to have adapted to local climatic conditions, but the adaptation of their pathogenic associates is poorly understood. Heterobasidion parviporum causes root and butt rot in spruce. In this work, the respiration of H. parviporum subpopulations from climatically diverse environments was examined at various temperatures. Isolates were obtained from three areas in Europe (southern Finland, Denmark and northern Italy) and from two locations in Central Siberia (Krasnoyarsk and Irkutsk). Respiration rates were measured in gas tight vials at eight temperatures from 0 to 33°C, using spruce saw dust as the sole substrate. Strains from Siberian locations with cold winters had higher activity at low temperatures (2–15°C) than strains from European locations with mild winters. Respiration rates of Siberian subpopulations increased more than those of European strains when the temperature rose from 0 to 6°C, but the increase was greater with the European subpopulations when the temperature increased further from 6 to 20°C. Only small differences were found among European as well as Siberian subpopulations. Variation in respiration rates between subpopulations was low compared to variation within subpopulations. Using strains isolated 2–18 years ago and thereafter stored at 5°C, we found lower respiration rates at 20°C in older isolates, independent of geographical origin, suggesting phenotypic plasticity of H. parviporum in regard to responses to temperature. Based on these findings, we propose that subpopulations of H. parviporum have the potential to adapt to global warming.     

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.     

Extent of colonization by Raffaelea quercivora of artificially inoculated living and gamma‐ray‐sterilized seedlings of two Japanese and three American oak species

Mass mortality of Fagacean tree species caused by Raffaelea quercivora has occurred widely in Japan. Because conidia or other propagules of the pathogen have not been found in infected trees, pathogen spread is assumed to occur primarily by hyphae. To clarify the relationship between hyphal growth of the pathogen within trees and their vessel arrangements, we examined two native Japanese oaks, Quercus crispula and Quercus glauca, and three exotic American oaks, Quercus coccinea, Quercus palustris and Quercus rubra. Quercus glauca is a radial‐porous species, whereas the other four species have a ring‐porous wood structure. Hyphal growth within inoculated potted living seedlings and in cut, sterilized stem segments of these species was examined microscopically after fungal inoculation. Water conductance in the seedlings was examined using transverse stem sections. The proportion of non‐conductive sapwood in Q. crispula, Q. coccinea and Q. palustris differed between inoculation and control treatment, being much higher in inoculated seedlings. The proportions were positively correlated with the extent of the hyphal growth. In sterilized stem segments, the extent of fungal colonization varied among the foreign ring‐porous species Q. coccinea, Q. palustris and Q. rubra. It is hypothesized that the extent of colonization by R. quercivora reflects the extent of non‐conductive sapwood irrespective of tree species, but is little affected by vessel arrangements.     

Presence and viability of Ceratocystis lukuohia in ambrosia beetle frass from Rapid ʻŌhiʻa Death‐affected Metrosideros polymorpha trees on Hawaiʻi Island

Rapid ?ōhi?a Death (ROD) is a fungal disease of ?ōhi?a lehua (Myrtaceae: Metrosideros polymorpha) caused by Ceratocystis lukuohia and C. huliohia. ROD is the aetiological agent of widespread mortality of this important tree on Hawai?i Island, but its epidemiology remains unclear. We investigated the prevalence and viability of C. lukuohia in ambrosia beetle frass in ROD‐affected ?ōhi?a trees. A total of 200 frass traps were placed onto C. lukuohia‐infected ?ōhi?a at four locations on the east side of Hawai?i Island. Frass was collected and screened for the presence of C. lukuohia DNA using a diagnostic qPCR assay. In addition, frass samples were screened for viability by carrot baiting. All trapped beetles were of the genus Xyleborus, with the majority being the non‐native X. ferrugineus. Of the frass samples tested, 62% contained C. lukuohia DNA and 17% of carrot baits were positive for the fungus. These results indicate that ambrosia beetle frass releases C. lukuohia into the environment. We discuss the potential role infested frass could play in the ROD pathosystem.     

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.     

The long‐term survival of Phytophthora cinnamomi in mature Banksia grandis killed by the pathogen

The ability of Phytophthora cinnamomi to survive long dry periods is the key to its persistence in the south‐west of Western Australia. It has been proposed that dead Banksia grandis are a significant long‐term reservoir for P. cinnamomi inoculum. To test this, 36 healthy B. grandis trees were inoculated in April 1999, and the presence of viable propagules in planta was determined between 2 and 34 months after tree death. By 10 months after inoculation, 75% of the trees had died, with the remaining seven trees dying by 22 months. The pathogen was more commonly recovered from bark than from wood, except from those trees that died at 22 months, and more commonly from above‐ground trunks than below‐ground trunks and roots until 8 months after plant death. In trees that died 12 months after inoculation, P. cinnamomi was recovered from 60% of trunk and root core samples at 3 months, declining to 33% at 10 months, 5.5% at 12 months and 0.1% at 34 months after tree death. In trees that died at 22 months, P. cinnamomi was recovered from 87% of trunk and root samples 2 months after tree death, decreasing to 0.5% by 33 months. This study suggests that the pathogen does not have a saprotrophic phase within dead B. grandis tissue, and B. grandis is unlikely to be a long‐term reservoir for P. cinnamomi. However, the manipulation of the density of B. grandis and the use of fire to facilitate the breakdown of dead Banksia trunks in the Eucalyptus marginata (jarrah) forest may reduce the spread and impact of P. cinnamomi.     

Root‐colonizing ophiostomatoid fungi associated with dying and dead young Scots pine in Poland

Ophiostomatoid fungi are carried by various bark beetles. However, very little is known about the role of these fungi in conifer roots. We studied ophiostomatoid fungi in roots of dying and dead Pinus sylvestris trees and tested the potential phytotoxicity of some isolates using a sensitive bioassay with Lepidium sativum in Poland. Fungi were identified based on their morphology and DNA sequencing. Three ophiostomatoid fungi, Leptographium procerum, Sporothrix inflata and Ophiostoma pallidulum, were isolated from the roots. The most abundant soil‐borne fungus, S. inflata, and relatively rare O. pallidulum were isolated for the first time from roots of dying and dead pine trees. The frequency of S. inflata and O. pallidulum correlated with tree decline. The fungi were isolated more frequently from roots of dead than dying trees. Sporothrix inflata and O. pallidulum slightly reduced the stem and root growth of L. sativum. Leptographium procerum reduced more significantly root than stem growth. This species reduced root elongation 32–54% after 10–17 days of incubation.     

Fungal colonization patterns in necrotic rootstocks and stem bases of dieback‐affected Fraxinus excelsior L.

European ash (Fraxinus excelsior) trees currently face the major threat of ash dieback caused by an invasive fungus, Hymenoscyphus fraxineus. Collar rots in F. excelsior have been increasingly associated with infections by this pathogen. However, the aetiology of the collar rots is still unclear and remains heavily debated. In contrast to most studies of this kind, entire rootstocks of four diseased ash trees were dug out to examine necrotic tissues in these rootstocks and stem bases in detail and to sample necrotic wood for fungal isolation. With the aid of morphological and molecular identification techniques, five to twelve fungal taxa were detected per tree. Members of the Nectriaceae family and Botryosphaeria stevensii, the causal agent of stem and branch cankers on many tree species, were frequently isolated from outer xylem. In contrast, H. fraxineus was the dominating species in interior wood layers. Microsatellite genotyping of 77 H. fraxineus isolates helped to identify up to six different genotypes per tree. The role of H. fraxineus and other isolated fungi in the aetiology of ash collar rots are discussed.     

Types of frass produced by the ambrosia beetle <Emphasis Type="Italic">Platypus quercivorus</Emphasis> during gallery construction,and host suitability of five tree species for the beetle

We studied the biology of the ambrosia beetle Platypus quercivorus in the logs of five tree species to determine: (1) the relationship between the amount of frass produced by beetles and tunnel length, (2) the relationship between frass shape and the morphological characteristics of P. quercivorus mouthparts, and (3) the suitability of five tree species for P. quercivorus reproduction. Five logs each from healthy Quercus crispula, Q. serrata, Castanea crenata, Sorbus japonica, and Cryptomeria japonica trees were used in this experiment. The results showed that there was a linear relationship between the amount of frass and tunnel length. Whenever powdery frass was produced, larvae were found in the gallery in the log, while fibrous frass was present only in galleries that contained just adults. The mouthparts of adults were completely sclerotized, which likely accounts for the fibrous frass production. Host preference of P. quercivorus was examined at two stages using five tree species. The first stage is digging initiation, which concerns male preference for digging a tunnel. More holes were made by males on S. japonica and Q. serrata logs, while fewer holes were made on C. japonica logs. The second stage is characterized by female orientation, mating, and progeny development. Platypus quercivorus could complete its lifecycle only in the two Quercus spp.