Variation in properties of Phlebiopsis gigantea related to biocontrol against infection by Heterobasidion spp. in Norway spruce stumps

The properties of 64 heterokaryotic strains of Phlebiopsis gigantea, isolated mostly from Norway spruce stumps, were tested for asexual spore production, growth rate and competitive ability against Heterobasidion spp. on agar medium, and for growth rate in spruce wood. Eighteen isolates were also tested for the efficacy of control against Heterobasidion spp. in stem pieces of spruce. The results revealed high variation in traits between different P. gigantea isolates. The efficacy of control against Heterobasidion spp. and the growth rate in spruce wood were closely related to each other (r = 0.727, p < 0.001). These preliminary tests indicate that the growth rate of a P. gigantea strain in spruce wood is the most important characteristic determining its efficiency in controlling the infection and spread of Heterobasidion spp. in spruce stumps.     

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.     

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.     

Presence of Heterobasidion infections in Norway spruce stumps 6 years after treatment with Phlebiopsis gigantea

Natural colonization by the root and butt rot causing fungi Heterobasidion spp. on Norway spruce (Picea abies) stumps following thinning and treatment with the biological control agent Phlebiopsis gigantea was investigated on three sites in southern Sweden 6 years after treatment. The fully treated stumps and control stumps were excavated and sampled to compare the survival of Heterobasidion spp. in the long term. Six years post‐treatment, 47 and 11% of untreated and treated stumps, respectively, had Heterobasidion infection. There was no difference in the relative infected area in discs collected from the butt and the roots for the different treatments. Control efficacy was 83% for treated stumps. After 6 years, there were no apparent differences between the remaining infections in treated compared with those in untreated stumps regarding the number of colonies, their size or relative infection area. Although infections, 3 months after treatment with P. gigantea, were significantly fewer and smaller than in untreated stumps, Heterobasidion inoculum can survive for at least 6 years in the stump and, when it does, constitute a risk for neighbouring trees.     

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.     

Stump treatment against Heterobasidion with Phlebiopsis gigantea and some chemicals in Picea abies stands in the western Alps

The effectiveness against Heterobasidion natural airborne infections on Norway spruce stumps was tested for six treatments: aqueous suspension of Phlebiopsis gigantea oidiospores, 10%, 20% and 30% aqueous urea solutions, 4% copper oxychloride aqueous solution, and borax powder. The experiment was carried out in four naturally regenerated forests in the western Alps, each characterized by a different airborne inoculum potential. Within each stand, all treatments, with the exception of urea 10%, were effective and resulted in colonized areas of stumps significantly lower than controls. The effect of ambient spore loads on the stump colonization is discussed.     

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.     

Biological control of Heterobasidion annosum on Pinus pinea by Phlebiopsis gigantea

The efficacy of a native isolate of Phlebiopsis gigantea in protecting the stumps of Pinus pinea against spore infection by Heterobasidion annosum s.str. was investigated. In preliminary tests carried out in stem pieces of P. pinea in confined environment, the efficacy of the isolate was compared with Rotstop^®, the commercial formulation prepared from a north European P. gigantea strain. Both showed a fully protective effect against artificial inoculation of H. annosum. Moreover, in stump treatment experiments carried out in a coastal P. pinea forest close to Rome, the native P. gigantea was effective against heavy airborne inoculum of H. annosum. Some natural infection by P. gigantea occurred in the stumps, but it was unable to control the pathogen. Random amplified microsatellite analysis allowed to recognize the presence of different P. gigantea strains in the forest.     

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.     

Occurrence of Heterobasidion basidiocarps on cull pieces of Norway spruce left on cutting areas and in mature spruce stands

Fruiting of Heterobasidion on cull pieces and stumps of Norway spruce was investigated in cutting areas and mature spruce stands located in southern Finland. Cull pieces of variable size and showing butt rot were left on three clear‐cut areas and in one thinned stand. Additionally, a part of the cull pieces was transported to mature forest sites with closed canopy. During the succeeding 3–4 years the cull pieces were investigated annually for sporocarps of Heterobasidion, and the area of actively sporulating pore layer of each sporocarp was measured. Root bases of spruce stumps in the logging areas were excavated and sporocarps found on the stumps also measured. At the onset of the experiment, Heterobasidion spp. were isolated from 76% of the cull pieces showing butt rot; 85% of the isolates were identified as H. parviporum and 15% as H. annosum s.s. During the following 3–4 years sporocarps were found on 20% of the 1938 cull pieces where Heterobasidion butt rot was initially detected visually. Sporocarp formation was promoted by advancement of butt rot, increasing cull piece diameter and end‐to‐end ground contact, but restricted by the colonization of the cull piece by Stereum sanguinolentum. Between‐site differences were significant but could not be explained by differences in tree cover. At the end of the investigation period the average sporulating area of Heterobasidion sporocarp per metre of cull piece was higher than the average sporulating area per stump at three of four logging sites. Hence, leaving cull pieces containing Heterobasidion butt rot at logging areas in southern Finland can considerably increase local production of Heterobasidion spores.     

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.     

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.     

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.     

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.     

Sensitivity of Root Rot Antagonist Phlebiopsis gigantea spores to high temperature or pressure

Aqueous suspensions of Phlebiopsis gigantea oidiospores, used as a biological control agent against Heterobasidion annosum, were either (1) set under pressure, (2) warmed or (3) used in mechanized stump treatment. Survival of P. gigantea following these treatments was monitored by estimation of oidia germination on agar medium and compared with the germination of oidia from control suspensions. Pressure up to 2200 kPa had no effect on the survival of P. gigantea. No effect on the survival was detected following incubation at ambient pressure for between 5 min and 8 h at 20°C. Phlebiopsis gigantea spore germination had an optimum at 30°C. When exposed to temperatures exceeding 30°C, viability of P. gigantea spores decreased with time. During mechanized stump treatment, survival of P. gigantea was not significantly lower, neither after the suspension had passed through the stump treatment device nor after 7 h time, compared with a fresh control suspension. Thus, mechanized application under normal summer conditions in Sweden did not obstruct the spore germination of P. gigantea in this study.     

Inoculation of pine stumps with Peniophora gigantea by chainsaw felling

By adding oidia of Peniophora gigantea to the lubricating oil of a chainsaw, stumps were automatically inoculated as the trees were felled. An experiment on Pinus sylvestris showed that direct chainsaw inoculation gave comparable results in terms of abundance of P. gigantea and control of Fames annosus, as conventional inoculation of stumps after felling.     

The efficacy of selected biological and chemical control agents against Heterobasidion abietinum on Abies cilicica

The efficacy of 30% aqueous urea solution, borax powder and spore suspensions of Phlebiopsis gigantea and Trichoderma harzianum against establishment of artificially inoculated Heterobasidion abietinum on Abies cilicica was tested both in the field and in a simulated stump treatment experiment carried out in a growth chamber. In the field, in each of the four selected stands 20 fresh stumps per control agent were treated and 20 stumps were left as untreated controls. In two of the stands, the treatments were applied in June and in the two others in November. Stumps were sampled twice, at 6‐ and 12‐months after treatment. In the growth chamber, 10 log pieces per treatment and control were used, and sampling performed after 6‐week incubation. Results of the urea and borax treatments were consistent between the experiments; the mean efficacies were 98.8 and 99.4% in the growth chamber, and 80.2–91.3 and 89.4–90.1% in the two samplings of the field experiment, respectively. Despite the high efficacies of the P. gigantea (85.9%) and T. harzianum (97.5%) treatments in the growth chamber, efficacies of these biological control agents in the field were 47.1–49.2 and 61.3–65.5%, respectively.     

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.     

Two studies to assess the risk to Pinus sylvestris from Heterobasidion spp. in southern Sweden

Abstract

Pine stumps are not being treated against Heterobasidion spp. in Sweden. To determine whether they should be, the frequency of stump infections and the species of Heterobasidion involved were investigated in nine newly thinned pure Scots pine (Pinus sylvestris L.) stands in southern Sweden. The incidence of Heterobasidion was measured in roots of standing Scots pine in another 15 stands. Infections by both H. annosum (Fr.) Bref. s.s. and H. parviporum Niemelä & Korhonen were numerous in stumps six months after a summer thinning. The pathogen, mostly H. annosum, was found in 44 of 60 sampled root systems, from 14 of the 15 stands. Twenty of the infected pines were assessed as healthy on the basis of crown symptoms while 24 trees had defoliated crowns. Infected root systems were most frequent among trees with thin crowns in stands on former agricultural land, where previous thinnings had been carried out during the growing season when airborne spores are plentiful. The study suggests that stump treatment may be a profitable way to reduce disease development in Scots pine monocultures on sandy soils as well as in mixtures with Norway spruce [Picea abies (L.) Karst.] on any soil.     

Terpene response of Picea abies and Abies alba to infection with Heterobasidion s.l.

The monoterpene composition of Picea abies and Abies alba resin was analysed in relation to growth by Heterobasidion spp. Fifteen‐year‐old P. abies and A. alba trees were inoculated on branches with three species of Heterobasidion annosum s.l. After 4 months of incubation, each host was colonized to a significantly greater degree by the pathogen specific to that host (H. parviporum on P. abies, H. abietinum on A. alba) than by the other fungi. Analysis of the enantiomeric monoterpene profiles in the spruce and fir showed that the response in terms of the relative proportions of the monoterpene compounds in the resin differed between tree species. Following challenge with Heterobasidion spp., A. alba trees did not show changes in monoterpene composition in addition to those in the wounding response (increase in (−)‐α‐pinene and (−)‐camphene, and decrease in β‐phellandrene). In P.abies, (−)‐α‐pinene, (+)‐α‐pinene and δ‐3‐carene increased following Heterobasidion attack but not after wounding alone.