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.     

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.     

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.     

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.     

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.     

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.     

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.     

Susceptibility to Armillaria novae‐zelandiae among clones of Pinus radiata

Options are currently limited for the management of armillaria root disease in plantations of Pinus radiata in New Zealand. One possibility may be to plant genetically resistant clones on infested sites. Studies were undertaken over four consecutive years to examine variation in inherent resistance in P. radiata. Rooted cuttings in pots were treated with wood segment cultures of Armillaria novae‐zelandiae and disease symptoms were monitored during the following summer. Disease was severe among inoculated cuttings in all studies, with an overall mean of 54% plants infected. There were significant differences in both infection and mortality between studies and between four pathogen isolates, but not among 25 radiata pine clones. The rate at which symptoms first appeared also did not vary significantly between clones. Results suggest that while genetic resistance cannot be ruled out as an option in P. radiata, potential gains may be limited in extent and costly to achieve.     

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.     

In vitro interactions in artificial and wood‐based media between fungi colonizing stumps of Sitka spruce

Interspecific interactions between fungi that colonize stumps of Picea sitchensis in Scotland were tested in dual cultures on Norkrans agar, spruce sawdust and in autoclaved blocks cut from roots. Isolates were ranked according to competitive ability on the different media, based on their ability to overgrow competitors or to form deadlock interactions. On the defined medium, Phaeolus schweinitzii was the species most able to overgrow competitors, followed by Stereum sanguinolentum and Heterobasidion annosum; Resinicium bicolor was the least able to overgrow competitors on this medium. By contrast, R. bicolor was the most competitive on spruce sawdust medium. Deadlocking interactions were formed most often in dual cultures on Norkrans agar. Observation of hyphal interactions on Norkrans agar under the microscope identified several different response types including growth of thin hyphae compared to control cultures, hyphal coiling, vacuolation of hyphae, hyphal lysis of one competitor and deposition of crystals in the agar. Hymenomycetes caused varying amounts of decay in autoclaved root blocks. Resinicium bicolor was able to replace other species in most co‐inoculations. Stereum sanguinolentum appeared to be the least competitive species in root block inoculations, being replaced by Melanotus proteus and R. bicolor, although interactions with H. annosum varied widely. These results indicate that substrate has a marked effect on interspecific fungal interactions, with wood‐based, particularly intact woody tissues closely matching competitive behaviour displayed in the field.     

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 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.     

Modern aspects of biological control of Fomes and Armillaria1

Biological methods can help to control root-infecting fungi such as Fomes annosus and Armillaria mellea. For example, inoculation of pine stumps with the fungus Peniophora gigantea is widely used against F.annosus in Britain. Experiments arc in progress to determine whether similar methods can be used for stumps of other conifers. The main sources of infection by Armillaria are stumps of broad-leaved trees, which are often treated chemically to prevent regrowth. Some of these treatments promote colonization by saprophytic fungi that compete well with Armillaria. In experiments, direct inoculation of stumps combined with chemical treatment has given encouraging results.     

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.     

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.     

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.     

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.     

Can Hymenoscyphus fraxineus infect hardy members of the Oleaceae other than ash species?

The interaction between the ash dieback fungus, Hymenoscyphus fraxineus, and two commonly grown members of the Oleaceae (Forsythia × intermedia ‘Lynwood’ and Ligustrum vulgare) was investigated. Shoots were inoculated with three isolates of H. fraxineus and harvested after 2 and 10 weeks. DNA was extracted from tissue collected at the point of inoculation, as well as 15 mm below and above (distal), and analysed using fungal‐specific ITS primers and specific primers for H. fraxineus. The fungus was frequently detected in tissue at the point of inoculation, but rarely in distal samples. There was no evidence that H. fraxineus was pathogenic on either of the two oleaceous species over the duration of the experiment.     

Susceptibility of common tree species in Sweden to Phytophthora cactorum,P. cambivora and P. plurivora

In Sweden, invasive Phytophthora pathogens have been recognized as a growing threat to urban and production forests, calling for an urgent update of regeneration strategies for infested areas. Stem inoculation tests were performed to test the relative susceptibility of common conifer and broadleaved tree species Pinus sylvestris, Picea abies, Larix x eurolepis, Betula pendula, Quercus robur, Fagus sylvatica, Populus trichocarpa and Tilia cordata to the root pathogens Phytophthora cactorum, P. cambivora and P. plurivora commonly isolated from Swedish soils. Results indicate that all the species tested were susceptible and formed lesions following stem inoculation with all three Phytophthora species, but to varying degrees. Of particular interest are the high levels of susceptibility in P. trichocarpa to all three Phytophthora species compared to other tested tree species.     

Effect of rhizobial inoculation on growth of <Emphasis Type="Italic">Calliandra</Emphasis> tree species under nursery conditions

A nursery experiment was conducted in un-sterilized soil in Senegal using six Calliandra species or provenances inoculated with a mixture of seven rhizobial strains. Plant growth was assessed periodically at 1, 2, 3, 12 and 18 months whereas nodulation, shoot and root dry weights were assessed at 12 and 18 months after planting (MAP). Un-inoculated seedlings of all the six species died at 12 months after planting. Results of growth assessments were variable with significant differences (P < 0.05) between C. calothyrsus Meissn provenances (Flores and San Ramón) and C. juzepczukii Standley in height at 1, 2 and 3 MAP. Shoot dry weights of San Ramón provenance of C. calothyrsus Meissn were also significantly different from those of C. acapulcensis (Britton and Rose) Standley and C. longepedicellata (Mc Vaugh) H. Hern and Macqueen at 18 MAP. In contrast, C. glandiflora (L’Her.) Benth grew poorly and did not nodulate. Although rhizobial inoculation improved shoot and root dry weights some differences were observed among the Calliandra species in response to the inoculation, which suggested the occurrence of interaction between the rhizobial strains and the host plant species tested.