Relationship between stump treatment coverage using the biological control product PG Suspension,and control of Heterobasidion annosum on Corsican pine,Pinus nigra ssp. laricio

The relationship between the proportion of the stump surface covered by the biological stump treatment agent PG Suspension, containing Phlebiopsis gigantea and its efficacy against the pathogen Heterobasidion annosum sensu stricto was studied during a first thinning of Corsican pine (Pinus nigra ssp. laricio) in Thetford Forest, UK. PG Suspension was manually applied to 100%, 75%, 50% or 0% of the surface of 150 stumps. Spores of H. annosum were inoculated onto 75 of the stumps, and the remaining stumps exposed to natural airborne spore deposition. The relationship between coverage and efficacy was found to be quantitative. Covering all the stump surface with PG Suspension completely excluded the pathogen, whereas stumps not treated with PG Suspension (the 0% treatment) became infected with H. annosum. Partial (75%) PG Suspension coverage resulted in the pathogen colonizing 40% of stumps following artificial inoculation with H. annosum, and just 7% of stumps exposed to ambient H. annosum spore infection. Decreasing levels of coverage allowed increasing areas of the stump surface to be colonized by H. annosum. Some small gaps in coverage were closed by lateral growth of P. gigantea, but it is recommended that operators aim for full stump coverage to give complete protection against H. annosum.     

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.     

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.     

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.     

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.     

Heterobasidion annosum infection of Picea abies following manual or mechanized stump treatment

Heterobasidion annosum (Fr.) Bref. s. l. colonization following thinning was studied in 1246 stumps of Picea abies (L.) Karst. trees from 14 previously unthinned stands in Sweden. Treatments included mechanized and manual application of (1) 35% urea solution, (2) Phlebiopsis gigantea (Fr.) Jül. spores, and (3) 5% solution of disodium octaborate tetrahydrate (DOT), and untreated stumps, cut in the summer and winter. Compared with untreated stumps cut in the summer, all stump treatments and winter cuttings significantly reduced the colonized stump area 6–7 weeks after thinning by 88–99%. Mechanized stump treatment provided as good protection as manual treatment against H. annosum infections. The probability of spore infection (p _ij ) was reduced by 53–83% in mechanized treatment and 79–98% in manual treatment compared with untreated summer thinning. In terms of p _ij , urea had significantly higher control efficacy than P. gigantea and manual treatment performed better than mechanized treatment.     

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.     

Untersuchungen zur biologischen Bekämpfung von Heterobasidion annosum an Fichte (Picea abies) mit antagonistischen Pilzen II. Interaktionstests auf Holz

Investigations on biological control of Heterobasidion annosum in Norway spruce with antagonistic fungi. II. Interaction experiments in wood . Seventeen fungal species were examined for antagonism against H. annosum in wood. After inoculation of stem sections and stumps by conidia and dowels, the distribution patterns of the mycelia within the wood were recorded. In spruce-wood antagonism was shown only by Hypholoma capnoides, Bjcrkandera adusta, Resinicium bicolor and Trichoderma spp. near the site of inoculation. As the distance from this site increased H. annosum became more dominant. Inoculations by dowels yielded more infections than inoculations by spores. The patterns of myeelial distribution within the wood were similar in stem sections and in stumps. Spruce stumps were colonized naturally mainly by Resinicium bicolor, Armillaria mellea s. I. and Nectria fuckeliana. In stem sections of Pinus sylvestris, however, Phlebiopsis gigantea displaced H. annosum effectively.     

Community of Aphyllophorales and Root Rot in Stumps of Picea abies on Clear-felled Forest Sites in Lithuania

The community of Aphyllophorales fungi in stumps of Picea abies (L.) Karst. and the occurrence of root rot caused by Heterobasidion annosum were investigated at 38 clear-felled sites in Lithuania. Fruit-bodies were recorded on 36.0% of the 3924 examined stumps. The most common were Bjerkandera adusta, Phlebiopsis gigantea, Fomitopsis pinicola, Gloeophyllum sepiarium, Stereum sanguinolentum, Trametes zonata and H. annosum. All species occurred in stumps both with and without established root rot, but B. adusta, F. pinicola, G. sepiarium, T. zonata and Trichaptum abietinum occurred less often in stumps containing root rot, while H. annosum was encountered more often. Greater numbers of fruit-bodies were found on stumps cut in summer than on those cut in winter. The fruiting became most abundant 3-4 yrs after the trees were felled. The abundance of nearly all Aphyllophorales correlated positively with the stump diameter, and on larger stumps, fruit-bodies of several different fungi were more common. The number of stumps containing H. annosum root rot in different sites varied from 9.8% to 68.8%, and was 27.6% on average. The incidence of root rot correlated neither with the age nor with the density of the felled stand, but it correlated negatively with the proportion of deciduous trees within a stand.     

Dry weight loss of wood after the inoculation of Scots pine stumps with Phlebiopsis gigantea

Artificial inoculation of stumps with Phlebiopsis gigantea (preparation‘PgIBL’) against Heterobasidion annosum in Scots pine (Pinus sylvestris) stands on former agricultural lands in Poland is being performed throughout the year. The practical efficiency of the biological control appears to be influenced by the moisture content in stumps and roots. In experiment 1, dry weight loss of wood 3 months and 6 months after P. gigantea inoculation in laboratory was investigated in the stumps after salvage cutting (felling of dead and dying trees) and after thinning (routine cutting of trees) and compared with the decay of the artificially inoculated stumps under field conditions. It was found that 6 months after inoculation the dry weight loss of the samples was about 3%, 17% and 22%, respectively. In experiment 2, the decay of wood from horizontal roots collected after thinning, inoculated with P. gigantea in laboratory, was evaluated. Three months after the inoculation, the loss of dry weight wood was about 22–52%, depending on initial moisture of the roots.     

Infection of Picea sitchensis and Pinus contorta stumps by basidiospores of Heterobasidion annosum

Infection of Pinus contorta and Picea sitchensis stumps by basidiospores of Heterobasidion annosum is extremely variable, both within and between sites, but in general P. sitchensis stumps are less sus-ceptible than those of P. contorta. Measurement of the cross-sectional area occupied by H. annosum on each stump provides a more sensitive test of species susceptibility than assessment of the proportion of stumps infected. P. sitchensis stumps become infected on a variety of soils but there is evidence to suggest that infection may be reduced by high rainfall. In some infected stumps, H. annosum is confined to the lower stump tissues. Its absence from the upper portion of the stump may be due to replacement by other micro-organisms or, alternatively, physical conditions in the upper stump tissues may prevent its continued survival after infection has taken place. In both species, but more commonly in P. sitchensis, some stumps remain alive for at least two years after felling, particularly on peat soils, due to the presence of root grafts with neighbouring trees. Results for P. sitchensis suggest that infection occurs more readily in living stumps than in those which die rapidly after felling. The viability of H. annosum basidiospore suspensions can be determined more accurately and more rapidly on a selective agar medium than on conifer stem sections.     

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.     

Modelling the incidence of Heterobasidion annosum butt rots and related economic losses in alpine mixed naturally regenerated forests of northern Italy

A total of 2257 recently felled stumps were sampled from 22 mixed naturally regenerated forest stands in the Aosta Valley, western Italian Alps, and examined for Heterobasidion annosum butt rots. Disease incidence ranged from 6% to 71% depending on sites, and H. annosum accounted for 92% of the total number of diseased stumps. H. annosum incidence was significantly higher (Chi‐squared multiple comparison tests, p < 0.05) on Norway spruce (44%) than on the other tree species (silver fir = 18%, larch = 12% and Scots pine = 6%). Based on the information on the airborne inoculum composition of the fungus, all the three European species of H. annosum were present, with a variable frequency, depending on site. A partial least squares regression analysis showed that the relative abundance of Norway spruce and four variables describing the size of trees were the best predictors for the incidence of H. annosum butt rots. A model having these variables as predictors was developed (r² = 0.75; p < 0.001) and successfully validated on five additional forest stands. An estimate of the losses in yield and value of timber associated with the disease, i.e. direct losses, was also carried out in the forests included in the survey. The percentage of direct financial losses was either comparable or lower than the average level of disease incidence.     

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.     

Biological control of Fomes annosus by Peniophora gigantea1

Fomes annosus is the most important root pathogen in British forestry. In pine crops, F. annosus is often checked when stumps are naturally colonised by Peniophora gigantea. Experimental inoculation of stumps with P. gigantea reduced infection by F. annosus in first rotation crops and was as effective as chemical stump treatments. In severely diseased pine crops, P. gigantea inoculation reduced but did not eliminate F. annosus. P. gigantea is produced commercially and is used in many pine forests in Britain.     

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.     

Resinicium bicolor; a potential biological control agent for Heterobasidion annosum

After introductory competition tests in the laboratory, Resinicium bicolor was used as a potential control agent for the conifer root rot fungus Heterobasidion annosum. Greenhouse pathogenicity tests with R. bicolor on 4-year-old seedlings of Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) showed moderate incidence of infection. In three places in Sweden, four test areas were chosen for field experiments in first-rotation plantations and also in old forest sites of Norway spruce. Wood blocks, pre-inoculated with one strain of R. bicolor, were buried in the soil beside stumps at 0, 1, 2, 3 and 4 months after thinning using various spatial designs. In two of the test areas, half of the stumps were treated with a suspension of H. annosum conidia from one strain by surface spraying. After 2–3 years, stump roots were investigated and the length of growth of both species were noted. The identity of mycelia reisolated from and wood debris in the test areas were confirmed by somatic compatibility tests with the original strains. The strain of R. bicolor released was recovered from all over the test area; the released H. annosum strain was only reisolated from the conidia sprayed stump roots. R. bicolor had little effect on the growth and occurrence of H. annosum. Potential control of disease spread may arise, however, from occlusion of the pathogen from outer parts of roots.     

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.     

Controlling and predicting the spread of heterobasidion annosum from infected stumps and trees of picea abies

Two Norway spruce stands with heavy infections of Heterobasidion annosum were clear‐cut in 1957 and 1959 in Sweden. The stumps were extracted, the soil sifted to remove most of the roots, and young Norway spruce were planted. After 25 and 28 years, H. annosum had infected 1 % and 2% of trees on plots where stumps had been removed and 17 % and 12% of the trees on control plots, respectively. Several of the H. annosum clones fruiting on old‐growth stumps were also detected in decayed, standing trees. The same fungal clone was found to be infecting adjacent trees from several old‐growth stumps. In addition to old stumps, stumps from recent thinnings and diseased living trees were traced as infection sources. Their relative importance in spreading disease was estimated. Disease risk predictions based on the distance of a tree from various infection sources correlated well with observed frequencies of rot.     

Efficacy of different concentrations of Rotstop® and Rotstop®S and imperfect coverage of Rotstop®S against Heterobasidion spp. spore infections on Norway spruce stumps

Two Rotstop^® preparations, one containing a Phlebiopsis gigantea strain from Finland (Rotstop F) and the other one from Sweden (Rotstop S), were used in different concentrations to treat thinning stumps of Picea abies against spore infections by Heterobasidion spp. in southern Sweden. Trees were cut on three sites during the summer of 2004, and 285 stumps were treated manually ensuring 100% coverage. Spore concentrations in the treatment suspensions were ca 5 × 10⁶ and 10 × 10⁶ spores/l, and approximately 10 ml of suspension was applied per 100 cm² of stump surface. An additional 31 stumps on one of the sites were treated mechanically with Rotstop S; in this treatment the spore concentration was high, about 20 × 10⁶ spores/l, but the coverage was incomplete. Three months later there was a significant reduction in frequency and relative areas of Heterobasidion spp. infections on stumps with manual treatment compared with the untreated stumps. However, there was no significant difference between the preparations or the concentration of active ingredient in terms of their reduction of Heterobasidion infection. Mechanical treatment with incomplete coverage failed to control infection. Therefore, in conditions of moderate Heterobasidion spore load in the environment there seems to be no reason to use higher concentrations of P. gigantea in the treatment of spruce stumps.