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 wood moisture on infection of Sitka spruce stumps by basidiospores of Heterobasidion annosum

In an inoculation experiment to test the effect of wood moisture content on infection of Sitka spruce (Picea sitchensis) stumps by basidiospores of Heterobasidion annosum, exposure to high rainfall increased infection in the heartwood and reduced sapwood infection compared to covered stumps. This was associated with a greater moisture content in both wood types. Within-treatment variation in the amount of infection was high and it is suggested that endogenous factors in stumps may have a greater influence on infection than the environment or the availability of spores.     

Physical and chemical responses of Sitka spruce (Picea sitchensis) clones to colonization by Heterobasidion annosum as potential markers for relative host susceptibility

Forty-one 2-year-old clones of Picea sitchensis (Bong.) Carr. from three full-sib families (14 clones from each of two families and 13 clones from a third family) were either wounded and inoculated with an isolate of Heterobasidion annosum (Fr.) Bref. or wounded without inoculation. Lesion lengths on the inner bark from the point of inoculation varied among clones 35 days after treatment. There was no relationship between lesion length and relatedness within families. Two clones (21342 and 25202) with the shortest lesions, tentatively designated as less susceptible to H. annosum, and two clones (21176 and 27166) with the longest lesions, designated more susceptible, were selected for comparison of host anatomical and chemical responses to infection. The position and structure of the ligno-suberized boundary zone (LSZ) in the bark of the clones suggested that the less susceptible clones formed thicker layers of suberized cells in the LSZ following wounding plus inoculation. No LSZ was observed in two ramets of the more susceptible Clone 27166 following wounding and inoculation with H. annosum. Compared with more susceptible genotypes, clones of P. sitchensis with low susceptibility to H. annosum had high relative proportions of (+)-alpha-pinene, (-)-beta-pinene and one unidentified terpene constituent (Unknown-15) in cortical resin sampled 25 cm from the lesions. In contrast, more susceptible clones had higher relative proportions of (-)-limonene, Unknown-16, Unknown-18 and Unknown-19. In the secondary resin produced in bark tissues surrounding the lesions, proportions of several monoterpene constituents varied; these changes included a decrease in the relative amount of beta-phellandrene and corresponding small increases in some minor constituents. The concentrations of the monoterpenes, except a few minor constituents, increased in the infected tissues. Wounding plus inoculation with H. annosum resulted in varied monoterpene responses, with distinct differences between less susceptible and more susceptible clones. In less susceptible clones, Unknown-19 increased following wounding plus inoculation, whereas in more susceptible clones, concentrations of delta-3-carene and Unknown-13 and Unknown-16 increased. Differences in both constitutive and induced resin monoterpene profiles may provide useful markers for resistance to H. annosum in selection and breeding programs.     

Infection of roots of Norway spruce (Picea abies) by Heterobasidion annosum

Wounding of excised roots of Norway spruce [Picea abies (L.) Karst.] caused a rapid accumulation of phenolic compounds, decrease of starch content and of certain enzyme activities in the ray cells. Infection of the wounds by Heterobasidion annosum (Fr.) Bref. intensified these processes, caused oxidation of the phenols, and induced activity of acid phosphatase and β-glucosidase in the ray cells of the infected area. The reactions were oxygen-dependent. Intra- and extracellular enzymes were detected in the wood-infecting hyphae. Other wood-growing fungi caused similar reactions in the wood as H. annosum.     

Infection studies with Heterobasidion annosum on young trees of Picea abies

Infection studies with Heterobasidion annosum were carried out on 15-year-old, cloned spruces between 1971 and 1978. The inoculum was introduced into the sapwood of the test plants at the base of the stem. All inoculations, conducted at different times of the year, were uniformly successful. The pathogen spread through the xylem tissue rapidly during the vegetation period, but from early autumn onwards gradually disappeared over the following two years. After this period, the fungus could no longer be detected.     

Responses of Sitka spruce of different genetic origins to inoculation with Heterobasidion annosum: lesion lengths,fungal growth and development of the lignosuberized boundary zone

Three genetically distinct groups of Sitka spruce, open‐pollinated Queen Charlotte Island provenance, A13 selected seed and M0044 half‐sib mixture, were wounded alone or wounded and inoculated with Heterobasidion annosum sensu stricto on the lower stems. Growth of the pathogen and lesion formation was compared in the three genetic groups after treatment. No differences in the rate of colonization of the three genetic groups were observed over a 40 day period; lesion lengths in the bark and sapwood correlated closely. Moreover, lesions were considerably longer in inoculated plants than in those which were wounded only. No correlations were found within or between host genetic groups in the numbers or total areas of resin canals present in the first 18 mm from the wound in bark tissues for the three host genetic groups. Formation of the ligno‐suberized boundary zone (LSZ), however, was inhibited in the bark of inoculated plants, being first detected later and at a greater distance from the wound/inoculation point in the presence of H. annosum than in plants that were wounded only. Thickness of the suberin cell layers within the LSZ of M0044 plants was greater in wounded and inoculated, than in wounded only plants. The significance of these findings in relation to detecting spruce genotypes potentially resistant to H. annosum is discussed.     

Compartmentalization of decay associated with Heterobasidion annosum in roots of Pinus resinosa

Decay associated with Heterobasidion annosum was compartmentalized in roots of Pinus resinosa.     

Liberation of Heterobasidion annosum conidia by airflow

The effect of speed and humidity of the airflow and the míst sprayed with airflow on the liberation of conidia of Heterobasidion annosum from pure cultures was studied in a small windtunnel. The conidia were liberated more easily in humid than in dry airflow. Liberation was most abundant when mist was added to the airflow. Threshold speed for the liberation of conidia was calculated to vary between 1.8 m/s and 3.9 m/s. According to the results, conidia of H. annosum may be liberated into the air by wind gusts associated with high humidity or mist and thus the conidia may have a role in aerial dispersal, although this is probably small compared with basidiospores.     

Effectiveness of some biocontrol and chemical treatments against Heterobasidion annosum on Norway spruce stumps

The biocontrol agents Trichoderma harzianum, Verticillium bulbillosum, Hypholoma fasciculare, Phanerochaete velutina, Vuilleminiia comedens together with the chemical treatments propiconazole and the culture filtrate of V. bulbillosum were tested on Norway spruce stumps during 2 years in order to evaluate their effectiveness against Heterobasidion annosum. The results were compared, in 1 year, with the effectiveness of Phlebiopsis gigantea. All the treatments reduced H. annosum infections. The best results were obtained with P. velutina, propiconazole and with culture filtrates and conidial suspensions of V. bulbillosum.     

Response of Compensatory-Fertilized Pinus sylvestris to Infection by Heterobasidion annosum

The effects of compensatory fertilization on the infection and growth rate of Heterobasidion annosum were studied in a 40-yr-old, naturally regenerated Scots pine (Pinus sylvestris L.) stand in south-eastern Finland. The treatments were: (1) unfertilized control, (2) a compound fertilizer containing P, K, Ca, Mg, S, Cu, Zn and B, (3) as treatment 2 but with nitrogen, (4) as treatment 3 but with limestone, (5) a mixture of nitrogen and limestone composed on the basis of needle analysis, and (6) wood ash. Pine roots were inoculated on two occasions four and five growing seasons after fertilization using four different strains of the P type of H. annosum. Two years after inoculation, the fungus was alive in 28.9% of the 135 inoculated roots. Fungus survival was highest in the control trees (50%) and poorest in the treatments 2 and 3 (10.5% and 16.7%, respectively). These two treatments differed significantly from the control. Total mycelial extension during the 2-yr study period was 16.4, 2.1, 4.5, 18.1, 15.2 and 5.3 cm in treatments 1, 2, 3, 4, 5 and 6, respectively. Only treatment 2 differed significantly from the control (p<0.05, df=23). The result suggests that the application of slow-release compound fertilizers without supplementary limestone may increase the resistance of Scots pine to infection by H. annosum. The nitrogen-free compound fertilizer may also retard the spread of annosum root rot in infected pine stands.     

Effects of vitality fertilization on the growth of Heterobasidion annosum in Norway spruce roots

The effects of vitality fertilization on the growth of Heterobasidion annosum in roots of Norway spruce (Picea abies) were studied in a 53-year-old, naturally regenerated spruce stand in southern Finland. The fertilizer treatments were: (1), unfertilized control; (2), a compound fertilizer containing P, K, Ca, Mg, S, Cu, Zn and B; (3), as 2 with additional nitrogen; (4), as 3 with additional lime; and (5), a mixture composed on the basis of needle analysis, containing N, P, K and Cu. Three growing seasons after fertilization, four roots of eight trees in each treatment were inoculated with four different strains of H. annosum. Spread of the fungus from the inoculation point was determined after 12 months. Mean spread rates upwards in roots were 18.2, 25.6, 21.3, 26.0 and 29.8 cm/year in treatments 1, 2, 3, 4 and 5, respectively. These results suggest a tendency towards faster growth by H. annosum in fertilized trees. However, there was considerable variation in fungal growth at both the tree and root level and differences between treatments were not statistically significant.     

Immunocytochemical localization of pathogenesis-related proteins in roots of Norway spruce infected with Heterobasidion annosum

The occurrence and accumulation of β-1, 3-glucanase and chitinase in seedling roots of spruce (Picea abies) following challenge by the root-rot pathogen Heterobasidion annosum were studied. Chitinase activity increased 2–3 fold following inoculation, whereas no significant increase in the activity levels of glucanase was recorded during infection. With TEM immunogold labelling, the enzymes were localized in protein aggregates in host tissues and in the cell walls of intercellular hyphae. Gold particles were sparse and irregularly distributed within host-cell walls. Only minor labelling was observed on hyphal walls coated with electron-dense materials. The labelling intensity increased with infection time and was always higher than in non-infected seedling roots. When this experiment was repeated using root samples inoculated with the saprophyte Phlebiopsis gigantea, a similar labelling pattern was observed. The cross reactivity of antisera raised against sugar-beet chitinase and glucanase with spruceroot enzyme extracts was demonstrated using dot-blot assays and ELISA.     

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.     

In vitro inhibition of Heterobasidion annosum by Phaeotheca dimorphospora

The antagonistic fungus Phaeotheca dimorphospora has been tested in vitro on agar plates and wood discs to evaluate its potential use against P (Eastern Canada) and S (Finland) intersterility groups strains of Heterobasidion annosum using two methods. Production of antifungal metabolites by P. dimorphospora was demonstrated on agar plates as well as on Pinus resinosa and Picea abies discs using the bi‐layer technique. Diffusible metabolites inhibited the growth of the pathogen on both substrates. Direct confrontation on wood discs showed that a 7‐day period of advanced growth by P. dimorphospora completely prevented the colonization of the tissue by H. annosum. Control was partial when the antagonist had only one day of advanced growth. Growth inhibition was superior against S group strains with less than 1% of the samples colonized by H. annosum compared with 22% for P group strains. Moreover, P. dimorphospora was isolated in more than 83% of the samples in the absence of the pathogen in both wood species. In vitro tests demonstrated that P. dimorphospora is able to colonize red pine as well as Norway spruce tissue and to use wood components for the production of antifungal metabolites.     

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.