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

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

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.     

Evaluation of the biological control agent Rotstop in controlling the infection of spruce and pine stumps by Heterobasidion in Latvia

The biological control agent Rotstop^® composed of a suspension of spores of Phlebiopsis gigantea (Fr.) Jül. is widely used for protecting conifer stumps from aerial infection by Heterobasidion species. The efficacy of Rotstop application on Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) stumps was determined in several locations and at different seasons in Latvia. Mean efficacy in controlling natural infection by Heterobasidion spp. in spruce stumps was 64%, calculated on the basis of number of infected stumps, and 89%, calculated on the basis of area of infected wood on sample discs cut from the stumps. Corresponding proportions for pine were 82% and 95%. The results show that Rotstop can be successfully used for stump treatment in Latvia, although improved efficacy is desirable, particularly in spruce. A Latvian isolate of P. gigantea, selected from numerous isolates in preliminary tests, was included in one experiment and was shown to be as effective as the Rotstop isolate. In untreated spruce stumps Heterobasidion spp. and P. gigantea were present in the same stump three times more frequently than in untreated pine stumps. Heterobasidion spp. infection in untreated spruce stumps was low when P. gigantea covered more than 10% of stump dissection.     

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.     

Intersterilitätsgruppen und Klone von Heterobasidion annosum-Isolaten aus Koniferen- Wurzel- und -Stammfäulen

Intersterility groups and clones of Heterobasidion annosum isolates from root and butt rots of conifers 69 H. annousum heterokaryons from scots pine, Douglas fir, Norway spruce and larch belonged to the P group, three heterokaryons from Norway spruce to the S group. The results of a study with clones of H. annosum isolates from roots of Scotch pine trees in close neighbourhood suggest colonisation by H. annosum (partly) via root contacts.     

In vitro interactions between bacteria isolated from Sitka spruce stumps and Heterobasidion annosum

Culture medium composition affected antagonism by bacterial isolates from Sitka spruce (Picea sitchensis) stumps against Heterobasidion annosum. Fifty percent of bacterial isolates inhibited H. annosum growth on sporulation agar or yeast–dextrose–peptone agar; only 10% of isolates caused inhibition on both media. Proportions of isolates inhibiting H. annosum varied with stump age; fewer isolates from 4‐ or 6‐year‐old stumps exhibited antagonism than isolates from older or younger stumps. Fifteen isolates showing antagonism on sporulation agar were tested against H. annosum in spruce wood cubes. None of the bacterial isolates alone caused a significant weight reduction in inoculated cubes. Relative inoculation times of bacterial isolates and H. annosum had an effect on weight loss in interactions; simultaneous inoculation with isolates and H. annosum inhibited weight loss caused by H. annosum compared with bacteria‐free controls. Inoculation with bacterial isolates 10 days before H. annosum had no effect on the decay rate. In contrast, inoculation with H. annosum 10 days before bacteria increased weight loss of cubes by 200% relative to cultures lacking bacteria. The effect of a mixed bacterial inoculum on weight change in 0.2‐mm spruce wood slips co‐inoculated with H. annosum, Resinicium bicolor, Hypholoma fasciculare, Stereum sanguinolentum or Melanotus proteus differed between different fungi.     

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.     

The spreading of the S type of Heterobasidion annosum from Norway spruce stumps to the subsequent tree stand

The occurrence of Heterobasidion annosum in stumps and growing trees was investigated on 15 forest sites in southern Finland where the previous tree stand had been Norway spruce (Picea abies) infected by H. annosum, and the present stand was either Scots pine (Pinus sylvestris), lodgepole pine (Pinus contorta), Siberian larch (Larix siberica), silver birch (Betula pendula) or Norway spruce 8–53 years old. Out of 712 spruce stumps investigated of the previous tree stand, 26.3% were infected by the S group and 0.3% by the P group of H. annosum. The fungus was alive and the fruit bodies were active even in stumps cut 46 years ago. In the subsequent stand, the proportion of trees with root rot increased in spruce stands and decreased in stands of other tree species. On average, one S type genet spreading from an old spruce stump had infected 3.0 trees in the following spruce stand, 0.5 trees in lodgepole pine, 0.3 trees in Siberian larch, 0.05 trees in Scots pine and 0.03 trees in silver birch stand. Although silver birch generally was highly resistant to the S type of H. annosum, infected trees were found on one site that was planted with birch of a very northern provenance.     

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

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

Management of East England pine plantations affected by Heterobasidion annosum root rot

Heterobasidion annosum causes mortality and volume loss in first and second rotation pine crops. Control measures include stump treatment with Peniophora gigantea and stump removal on severely diseased sites.     

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.     

Effectiveness of treatment of Norway spruce stumps with Phlebiopsis gigantea at different rates of coverage for the control of Heterobasidion

The natural establishment of the root and butt rot causing fungus Heterobasidion annosum s.l. on Norway spruce (Picea abies) thinning stumps treated with Phlebiopsis gigantea was investigated on seven sites in southern Sweden. The trees were cut during summertime and the stumps were treated with different patterns simulating the effect of mechanical stump treatment with a single‐grip harvester. Sampling was conducted 3 and 12 months after treatment. At both samplings, the best control was obtained when 100% of the stump surface was covered by P. gigantea: in contrast, untreated control stumps showed the highest incidences of H. annosum s.l. infection at both sampling times. However, 30 and 26% of the fully covered stumps at the first and second samplings, respectively, were diseased, and question the efficacy of treating Norway spruce stumps with this biological control agent in Sweden.     

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.     

Susceptibility of Sitka spruce and grand fir trees to decay by Heterobasidion annosum

The susceptibility of Sitka spruce (Picea sitchensis) and grand fir (Abies grandis) to decay by Heterobasidion annosum was compared on a site with a previous history of serious disease. The incidence of decay 15–16 years after planting was 1.5% in grand fir, compared with 34.8% in Sitka spruce. These results are broadly similar to those reported from other trials. More information is required for older trees in Britain but the evidence so far available suggests that grand fir is more resistant to H. annosum than Sitka spruce. Planting grand fir on severely infested sites may therefore significantly reduce the serious losses that could be expected with Sitka spruce. In 14 out of 271 decayed Sitka spruce H. annosum was absent from the middle or base of the decay column but present at the top. Approximately 3% of infected spruce showed features suggesting some degree of resistance. Resin bleeding was observed at the base of 23.2% of the infected spruce, and the mean height of the decay column in these trees was significantly greater (0.96 m) than in those without this feature (0.66 m). The inoculum for infection was provided by stumps of the previous crop, and the genets of H. annosum that were present in some infected trees were also identified in stumps. The majority of infected trees contained only a single genet but three trees contained two genets. In this young crop individual genets were smaller than those found elsewhere; the largest extended to include six trees.     

Initial reactions in sapwood of Norway spruce and Scots pine after wounding and infection by Heterobasidion parviporum and H. annosum

The xylem surface of seedlings, stem material and roots of Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) were inoculated with strains of Heterobasidion annosum s. str. and H. parviporum s. str. The depth of necrosis in wounded spruce increased at a linear rate for at least seven weeks of incubation, but the rate of necrotic spread was significantly faster in infected wounds. In wounded pine the necrosis was maintained at a more superficial level for several weeks. Both spruce and pine sapwood were initially infected by hyphae of both species. In spruce, the hyphae advanced at a constant rate behind the necrotic front. On the contrary, after 1–2 weeks living H. parviporum hyphae were rare in pine rays. Heterobasidion annosum hyphae survived in pine rays, phloem and tracheids, despite a heavy accumulation of phenolics and resins and were able to penetrate into the sapwood at a linear rate although slower than infections in spruce. Histochemistry and quantitative estimates demonstrated that peroxidase activity was initially higher in spruce sapwood than in pine. Within three days of incubation, the activity in spruce sapwood disappeared concurrently with deepening necrosis. However, in pine, in both control and infected samples, there was a significant increase in peroxidase activity in the area surrounding the superficial necrosis, up to the wound surface and in the cambium and phloem around the wound. After wounding and infection, the content of soluble protein increased significantly in wood of older trees but not in seedlings. Infection resulted in an increased formation of lipophilic extractives in both spruce and pine but to a significantly greater degree in the latter, whereas the amount of hydrophilic compounds decreased in both. High‐performance liquid chromatography (HPLC) analyses of lipophilic extracts showed that inoculation of pine with the two species of Heterobasidion increased the amounts of pinosylvin, its monomethylether and several other phenolics as also resinous compounds. The results obtained may be relevant in explaining the known higher resistance of Scots pine to H. parviporum.     

Optimizing the management of a Picea abies stand under risk of butt rot

A simulation model was developed to predict the growth of a Norway spruce stand under risk of butt rot caused by Heterobasidion annosum stump infection and logging injuries. The simulation model was distance‐dependent; tree growth was predicted with a distance‐dependent model, and the spread of butt rot through root contacts depended on tree location. Infection of stumps and injured trees, and the spread of butt rot in the stand were stochastic processes whereas tree growth and mortality were treated as deterministic processes. The simulation model was used with the nonlinear optimization algorithm of ooke and J eeves (J. Assoc. Comput. Mach, 8, 212–229, 1961) to find the most profitable management schedule for an even‐aged, young stand. Optimization used four different stump infection rates and two spreading capacities from infected stumps. The profitability was evaluated by the expected soil expectation value (SEV) at a 3% interest rate. Two thinnings, both in winter‐time, and hence without H. annosum infections, resulted in the highest SEV. If any stump infection by H. annosum occurred, only one thinning and a shortened rotation were suggested. The optimal thinning rate tended to decrease but also large trees were removed with the increasing infection rate. With one thinning during a rotation, stump treatment was profitable above a stump infection rate of 10%     

Reaction zone and periodic increment decrease in Picea abies trees infected by Heterobasidion annosum s.l.

Norway spruce (Picea abies) trees infected by Heterobasidion annosum s.l. decrease their periodic increment after a long period of time. Periodic increment decrease hypothetically relates to the formation of a reaction zone in order to prevent fungal colonisation. We studied 11 stands in Sweden, where we compared the periodic increment of healthy, rotten- and H. annosum-infected trees growing on plots thinned in winter, unthinned or thinned in summer, with and without urea or P. gigantea treatment of the stumps. Based on the rot incidence and the population structure of H. annosum of the plots, two phases of infection were considered: > 13 years and < 13 years. The presence of reaction zone and decay was observed on wood cores extracted with an increment borer. Rotten and H. annosum-infected trees with reaction zones exhibited a lower periodic increment than healthy trees (13.0% and 12.5% losses in terms of diameter, respectively), while no differences were observed between healthy trees and rotten and H. annosum-infected trees without reaction zone. Our results support the hypothesis of a periodic increment decrease in individual trees due to photosynthate re-allocation resulting from decay compartmentalization. Periodic increment decrease was only evident in trees that had been infected for more than 13 years. Trees in urea-treated plots registered a higher periodic increment, suggesting a possible response of trees to the nitrogen addition of the urea treatment of the stumps.