Further Observations on Fungi Inhabiting Pine Stumps

Growth-rates, both on malt agar and in stump wood, of earlystump colonizers such as Fomes annosus, Pemophora gigantea,Stereum sanguinolentum, and Leptographium lundbergii are higherthan those of later-colonizing agarics. The initial invasionof stump roots usually proceeds via the stump body by fungiwhich infect the cut surface. Although stumps may dry out appreciably shortly after felling,in later stages of decay the moisture content of the wood oftenincreases considerably. Stump decomposition is associated witha succession of decay fungi, the ‘Peniophora’, ‘Hypholoma’,and ‘Tricholoma’ stages being distinguished. Severalphycomycetes and fungi imperfecti colonize decayed, but notfresh, stump wood. P. gigantea is shown to be a vigorous competitor of F. annosusfor initial colonization of stumps: it may also replace F.annosus in stumps. Gliocladium viride and Trichoderma viridecompete with wood-rotting basidiomycetes and sometimes replacethem in very decayed wood.     

The Infection of Pine stumps by Fomes annosus and other fungi

A study of the infection biology of stump-colonizing fungi hasshown that certain species, for instance Fomes annosus, Peniophoragigantea, Stereum sanguinolentum, and several causing blue-stain,initiate infection of fresh stumps by means of air-borne sporeswhich colonize the cut surface. The incidence of natural infectionis reduced considerably by applying creosote to the surfaceimmediately after felling. High resin content of stumps is correlatedwith increased resistance to infection. Marked seasonal variationin the incidence of stump infection by F. annosus and P. giganteawas observed. The spore content of the air, and competitionbetween fungi in stumps, are considered to be important factorsaffecting this variation. Initial colonization of stump roots usually proceeds from thebody of the stump. Invasion of roots by saprophytes presentin the soil or litter also occurs but only many months afterfelling. Crowns of plantation pines may bear many spores of stump-colonizingfungi, including F. annosus. Under the conditions described,the proportion of stump infections ascribable to rain-splash-bornespores was small compared with that caused by wind-borne spores.     

The Role of Host Vigour in the Susceptibility of Pines to Fomes annosus

The resistance of suppressed pines to F. annosus and in particularthe role of such trees as infection foci in disease-free standsis a matter of potential significance for the spread of thepathogen. Mycelial inoculations showed that, in contrast todominant trees, the roots of very heavily suppressed trees possessno active resistance to the spread of the pathogen. However,at this extreme stage of suppression root-surface-fungi canalso invade the roots and to a considerable extent replace thepathogen. Spore inoculations of small root wounds gave similarresults except that the rate of infection was very much slower. Mycelial inoculations with F. annosus, Leptographium lundbergii,Peniophora gigantea, and Trichoderma viride were carried outin the trunks of pines suffering from different degrees of suppression.The fungi differed in their adaptation to the host, L. lundbergiibeing the most specialized to the biological, chemical, andphysical characteristics of pine wood and T. viride the least.This adaptation was shown to be inversely related to the competitivesaprophytic ability of each fungus, including the ability toproduce and tolerate antibiotics.     

Culturable microfungi inhibitory to Armillaria rhizomorph formation from Fagus sylvatica stump roots and soil

Culturable fungi from 28 fungal communities were isolated from soil, rhizosphere and thick (1 cm diam.) roots of living beech (Fagus sylvatica) trees and their stumps 1–3 years after felling. All fungi were morphotyped and identified morphologically. The frequency of fungi was 2–5× greater in stumps than in living trees. The diversity of fungi was similar in living trees and stumps. The majority of fungal species that occurred at greater frequency on/in roots of stumps reduced the growth of Armillaria ostoyae and to a smaller extent of A. gallica rhizomorphs in a soil substrate in vitro. It is suggested that the mycobiota of roots may constrain the colonization of F. sylvatica by A. ostoyae rather than by A. gallica. The significance of these findings in the epidemiology of Armillaria in beech forests is discussed.     

Resin and the Resistance of Conifers to Fomes annosus

Roots of young trees of different conifer species, Scots pine,Corsican pine, Sitka spruce, and Douglas fir were inoculatedwith Fomes annosus. In the pines, infection was negligible butin the other genera the central wood was extensively invaded.The process of infection was studied further in Sitka spruceby means of a root-severing technique in which an inoculum blockwas forced between the cut ends of a root. In most trees a centralrot developed which in some extended up into the butt: the meanextent of growth recorded was 30 cm in 14 months. In the severedroot portions infection also occurred but F. annosus was subsequentlyreplaced to a considerable extent by other micro-organisms,particularly in trees growing on a peat soil. A similar centralrot developed after inoculations of Norway spruce. The resistance of pine sapwood and the outer wood of spruceand Douglas fir can be explained at least partially by the structureand viability of the resin duct system. Larch, Douglas fir,spruce, and pine form a series showing increasing complexityand activity of the resin system. In addition, studies carriedout on the resin yields of pines growing under different conditionsshowed that the resistance of a tree to F. annosus is correlatedwith its ability to mobilize resin. Site factors, forest management,host age vigour, genetic constitution, and past history mayall influence resin mobilization.     

Antagonism against fomes annosus. Comparison between different test methods in vitro and in vivo

The antagonism of Trichoderma harzianum, Scytalidium album and an unidentified fungus (isolated from Monotropa hypopitys) against the root rot fungus Fomes annosus was studied in vitro and in vivo. Antibiosis was demonstrated on malt extract agar and on powdered wood. Competition between F. annosus and its antagonists was studied in stem discs and in living stems of Norway spruce. Effective antibiotics on agar and sawdust did not always bring about effective antagonism against F. annosus in stem discs or living trees. Factors such as growth rate of the antagonists in wood and their ability to invade the substrate during competition may influence the effectiveness of F. annosus suppression. The results from studies on antagonism in stem discs agreed with the results from tests in living trees. The frequency of penetrations through the wood cell walls was drastically lowered by the presence of the antagonists but was not closely correlated with the abundance of F. annosus.     

Observations on the Biology of Fomes annosus, with Particular Reference to East Anglian Pine Plantations: III. Natural and Experimental Infection of Pines, and Some Factors affecting Severity of the Disease

Apparently Fomes annosus normally invades healthy, not damaged,roots, the wood being infected almost at once in mall roots,though in larger ones the fungus grows in the bark for sometime. Alkaline soils also favour the latter type of development.Infections of living roots are more frequent around infectedstumps in alkaline soils than in acid ones. Details are given of infection of pine roots induced experimentallyand of the factors affecting invasion. Greater resistance to killing is evinced by older tree and thoseon the margin of the plantation. Disease is leas severe in EastAnglia on acid soils and where there is more organic matteron the soil surface. These relations are apparently associatedwith the types of soil microflora (of which details are given),the antagonism of Trichoderme viride being especially important.     

Colonization of Fungi and Bacteria in Stumps and Roots of Scots Pine after Thinning and Treatment with Rotstop

The research areas were located in the Pisz Forest District, northeast Poland, in 10‐year‐old Scots pine (Pinus sylvestris L.) plantations, established in 2004 on a clear‐cut area. Reforestation was performed without a biological treatment against root pathogens, despite the presence of Heterobasidion annosum and Armillaria ostoyae in roots and stumps of trees growing previously. The aim of this research was to evaluate how thinning and treatment with the biological control agent Rotstop influences bacterial and fungal communities within roots and stumps. Twelve months after thinning, samples were collected from five stumps in each of two seasons, autumn and spring, from stands on two types of site, one previously forested and one agricultural (20 stumps in total). Wood samples were cultured on agar media, and (i) fungi in the upper part of the stump and (ii) in roots and (iii) bacteria in roots were genetically identified. Sequences were genetically identified by comparing sequences with records held in the GenBank database. We found great differences in the frequency of both fungi and bacteria in roots: they were more frequent (i) in healthy stumps compared to stumps infected with pathogens (H. annosum and A. ostoyae), (ii) in postagricultural soil than in forest soil and (iii) after spring rather than autumn biological treatment. The introduced species Phlebiopsis gigantea was only identified in the parts of the stumps which were above ground level. The bacterium Paenibacillus pini was associated with the presence of H. annosum infecting the stumps from the roots side. In areas seriously threatened by root pathogens, biological treatment can play only a limited role. It can spread to the upper part and impede the production of fruitbodies; however, it has no impact on the development of pathogens in deeper root areas.     

Impact of biological (Rotstop) and chemical (urea) treatments on fungal community structure in freshly cut Picea abies stumps

To control the infections by root rot fungi Heterobasidion spp., surfaces of freshly cut Norway spruce stumps are covered either by a biological (Rotstop; spore suspension of competitive saprotrophic fungus Phlebiopsis gigantea), or by a chemical (35% aqueous solution of urea) compound. In Fennoscandia, Rotstop and urea are applied, respectively, on 47,000 ha and on 2000 ha of forestland each year. The aim of this work was to assess the impact of biological and chemical control on biodiversity in communities of non-target fungi in freshly cut (7-week-old) stumps. Isolation of fungi to pure culture was accomplished from 402 wood samples taken from 63 stumps, 21 treated with each of the compounds and 21 untreated. The isolations yielded 368 distinct fungal strains representing 47 species. Stump treatment led to decrease of species richness both in Rotstop-treated (by 15%) and in urea-treated (by 19%) stumps. Nevertheless, the stumps subjected to the biological compound were colonized mainly by the same fungi that occurred naturally in untreated stumps (Sorensen similarity indices; S_S=0.69; S_N=0.68). By contrast, chemical treatment strongly promoted stump colonization by Ascomycetes and Deuteromycetes, led to significant decrease of Zygomycetes, and almost completely eliminated Basidiomycetes (including Heterobasidion spp.). Thus, resemblance to a natural community was low (S_S=0.45; S_N=0.34). Rotstop treatment decreased significantly the extent of stump colonization by Heterobasidion spp., and increased that of P. gigantea. All strains of the latter were genetically identical among themselves and to the Rotstop strain. The mechanisms of biological and chemical control, and biodiversity aspects are discussed.     

Changes in Microfungal Communities in Roots of Quercus robur Stumps and their Possible Effect on Colonization by Armillaria

Sixteen fungal communities were sampled by isolating from 0.5–1 cm diameter roots of living trees and stumps of common oak (Quercus robur). The density of fungi was 1.5–2 times greater in roots from stumps than from living trees. The diversity of fungi was similar in living tree roots and stumps. Some of the fungal species with increased densities in stump roots (e.g. Aspergillus kanagawaensis, Chrysosporium pannorum, Cylindrocarpon destructans, C. didymum, Hormiactis candida, Monodictys lepraria, Mycelium radicis atrovirens, Penicillium daleae, P. janczewskii, and Trichocladium opacum) usually stimulated the growth of rhizomorphs of either Armillaria ostoyae or A. gallica in oak‐wood segments in vitro. Eight of 27 isolates that were studied stimulated the rhizomorph growth in both Armillaria spp. It is presumed that the increase in density of `stimulants' may predispose oak stumps to infection by A. ostoyae and A. gallica.     

Changes in Sapwood of Roots of Norway Spruce, Attacked by Fomes annosus

Acetone extracts of sapwood and reaction zone of spruce roots attacked by Fomes annosus, collected in February, June and October, were separated into resinous and phenolic fractions. The fractions were further separated by column, thin layer and gas liquid chromatography, followed by biological tests, using Fomes annosus and other rot fungi. The reaction zone contained quantitatively less light petroleum soluble compounds than the sapwood but more acids. The phenolic content was about ten times higher in the reaction zone than in the sapwood. Nine lignans and one simple phenol (4-methylcatechol) were identified and quantitatively estimated in the reaction zone. The resinous fraction of the extract from the reaction zone as well as some of the lignans and 4-methylcatechol inhibited fungal growth, in some cases followed by detoxification and continued growth. The predominant lignan, hydroxymatairesinol, had no effect on Fomes annosus or five other wood degrading fungi. About 15 unidentified phenols were observed, some of them probably of importance as inhibitors, either alone or in combination with other phenols.     

The capacity in Heterobasidion annosum s.l. to resist overgrowth by the biocontrol agent Phlebiopsis gigantea is a heritable trait

The necrotrophic pathogen Heterobasidion annosum sensu lato (Fr.) Bref. causes severe root rot on coniferous trees in the boreal and temperate forests. The annual economic losses caused by this fungus in Europe are estimated to at least 790 million €. In managed forests, the major route of infection is via stump surfaces from which the H. annosum s.l. grows through the roots and attacks adjacent healthy trees. A biocontrol method to reduce H. annosum s.l. infection is to apply the wood degrading fungus Phlebiopsis gigantea in a spore solution (Rotstop) directly on the freshly cut stumps immediately after cutting. We investigated the potential risk for a build-up in the capacity of H. annosum s.l. to resist overgrowth by P. gigantea. Wood blocks of Picea abies, precolonized with the two fungal species, were juxtaposed on top of agar and the overgrowth of the P. gigantea strain (Rotstop) on the H. annosum s.l. was measured periodically. We found a natural variation in Heterobasidion parviporum to resist overgrowth by P. gigantea. There was no difference between homo- and heterokaryotic strains. In a mapping population of 91 progenies from a H. annosum hybrid strain we were able to identify one quantitative trait locus (QTL) which controls the examined resistance capacity. We estimated the broad sense heritability to 0.336 for the capacity to resist the P. gigantea overgrowth. We conclude that there exists a theoretical risk for resistance build-up in the H. annosum s.l. population towards its biological control agent P. gigantea.     

Natural shifts in communities of rhizosphere fungi of common oak after felling

Fungal communities in the rhizospheres of oak roots were shown to change after the trees were felled. The ecological significance of this is considered. Fungal communities were isolated from rhizospheres of thin roots (0.5–1 mm diam.) of living trees and their stumps two years after felling in 30- and 50-year-old stands of Quercus robur, with samples taken at two locations in each stand. In total, 128 species of fungi were isolated. Six species, Absidia cylindrospora, Penicillium adametzii, P. citrinum, P. daleae, P. janczewskii and Umbelopsis vinacea were detected in the rhizospheres of living oaks and stumps at all locations. The abundance of Geomyces spp.?+?Pseudogymnoascus roseus, Gymnoascus reessii and Zygomycetes was greater, and of Penicillium spp. was smaller, usually significantly, in rhizospheres of stump roots than of living tree roots. Among Zygomycetes, the populations of A. cylindrospora and Mucor hiemalis decreased, while those of Mortierella spp., particularly M. macrocystis, increased after felling. Species of Penicillium favoured by roots are P. adametzii, P. citrinum, P. daleae, P. herquei, P. janczewskii, P. raistrickii. On stump roots they are replaced by P. chrysogenum, P. islandicum and P. spinulosum. The combination of Chalara spp. with oak roots indicates the occurrence of specific relationships, suggesting that the organic food base provided by the plant is a primary factor determining the composition of the fungal community. Edaphic and other environmental factors may be less important in this effect.     

Root deformation in plantations of container-grown Scots pine trees: effects on root growth,tree stability and stem straightness

Root system deformation was studied in 23 Scots pine (Pinus sylvestris L.) stands in central Sweden. The study comprised both plantations created with container-grown plants (Paperpot) and natural stands including young (7–9 year old) and older (19–24 year old) trees. Trees were measured with regards to distribution of roots, root deformation, stability, stem straightness and wood properties in stumps. Root distribution was most uniform for naturally regenerated trees. Older trees generally showed a better root distribution than young trees. The young planted trees displayed a high frequency of severely spiralled root systems, while only a few of the older trees had spiralled root systems. No severe root deformations were observed on naturally regenerated trees. Naturally regenerated trees were more stable than those which had been planted. Differences in bending moment, when trees were pulled to an angle of 10°, were considerable between young planted and naturally regenerated trees, but less pronounced for the older trees. Young planted trees had the highest frequency of severely crooked stem bases, while naturally regenerated trees had the straightest mode of growth. Tensile strength in peripheral wood samples of the stumps was substantially lower for planted than for naturally regenerated trees. Strain values to breakage of wood samples, taken from the root collar and the central- and peripheral part of the stump were lower for planted trees. The conclusions from this study are that root distribution, tree stability and stem straightness of planted Paperpot-grown trees will improve after a certain time and approach the state of naturally regenerated trees. As trees grow older, early established crooked stem bases will be compensated by radial growth and the tree will appear straighter. Inside the stem, however, problems may still remain with abnormal fibre direction and compression wood together with inferior root strength due to fibre disturbances as a result of spiralled roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

The impacts of treatment with biocontrol fungus (Phlebiopsis gigantea) on bacterial diversity in Norway spruce stumps

The biocontrol agent Phlebiopsis gigantea has been intensively applied to the surface of Picea abies stumps to control Heterobasidion root rot. But little is known about the possible impact of this treatment on the resident bacteria community in the stumps. High throughput DNA bar-coded pyrosequencing was used to characterize the diversity of bacteria in the stumps of P. abies at 1, 6 and 13 years after treatment with P. gigantea. The sequences were classified into 12 phyla and 160 genera, of which Proteobacteria and Acidobacteria were the most abundant groups over time. Moreover, at the initial stages of decay, Proteobacteria were the most abundant whereas Acidobacteria were the most common at advanced stages of decay. Treatment with P. gigantea led to significant increase of the genus Acidobacteria-Gp1 at 1 year after treatment. The analysis of observed and estimated operational taxonomic units (OTUs) as well as diversity indices revealed that P. gigantea treatment significantly decreased the initial bacterial richness in the stumps, but the bacterial community gradually recovered and the negative effects of P. gigantea was attenuated. These results provide additional insight on the risk assessment as well as environmental impact on the long-term use of P. gigantea in the control of Heterobasidion root rot in conifer forests.     

Latent infections of <Emphasis Type="Italic">Fomes fomentarius</Emphasis> in the xylem of European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>)

Fungal colonisation originating from endophytic thalli in wood of healthy European beech trees (Fagus sylvatica) was studied. Fungi were isolated from wood immediately after felling and after incubation for 8, 16 and 24 weeks under two different drying regimes. Two media were used to isolate fungi: malt extract agar with and without thiabendazol. Thiabendazol was added to inhibit non-basidiomycetes. The two drying regimes had no influence on the species composition of the recovered mycobiota and the frequency of isolation of these species. Mycelia of basidiomycetes except Coniophora puteana emerged only from wood samples inoculated onto malt extract agar containing thiabendazol. Only a few isolates were obtained from freshly cut wood, but a great number of isolates was recovered already after eight weeks of wood incubation. Four taxa accounted for 88 % of the total number of isolates: Hypoxylon fragiforme, Trichoderma spp., and the basidio-mycetes Coniophora puteana and Fomes fomentarius. The latter had not been considered an endophyte before. The isolates of F. fomentarius were made exclusively from the stem and some large diameter branches, which are the locations of its basidiocarps on dying trees. Every isolate of F. fomentarius was genetically different as revealed by tests for somatic compatibility. Advantages of the presumed endophytic strategy of F. fomentarius are discussed. Microscopic investigations showed a great number of hyphae within the cell lumina of vessels and a distinct wood degradation already after eight weeks of wood incubation.     

Secondary metabolites and nutrients explain fungal community composition in aspen wood

Fungi are the main decomposers of litter and wood, driving carbon and nutrient cycles. Despite a large number of studies, fungal community composition is remarkably difficult to predict. In the present study, we explore the importance of secondary metabolites and nutrient content in wood and bark as determinants of fungal community composition. We used aspen (Populus tremula) logs of similar size, from one location, and measured concentrations of carbon, nitrogen and secondary metabolites in bark and wood sampled shortly after felling. Fungal DNA was extracted from logs directly after felling and after two seasons of decomposition, and the fungal communities were assessed using DNA-metabarcoding. Concentrations of metabolites varied considerably between individual trees, and we also observed significant differences within single trees. Plant metabolites and nitrogen concentrations significantly affected fungal community composition. For the overall fungal communities and for wood saprotrophic fungi, the explanatory power of wood and bark metabolites was highest in logs decomposed over two seasons. In recently felled trees however, concentration of metabolites had a stronger effect on plant pathogens and endophytes. We conclude that secondary metabolites represent an overlooked, but important niche dimension for fungal communities in both functional sapwood and dead wood.     

Role of Copper Binding, Absorption, and Translocation in Copper Tolerance of Agrostis gigantea Roth

The capacity of roots to accumulate and retain copper was examinedin two clones of Agrostis gigantea which differ in their toleranceto excess copper. Root elongation growth in the non-tolerantclone was completely inhibited by 16 mmol m^–3 Cu whereas40 mmol m^–3 was required for inhibition in the tolerantclone. The amount of readily exchangeable copper was greaterin roots of the tolerant clone than in the non-tolerant clone.The higher capacity for binding copper did not prevent the entryof copper into the cells of intact or excised roots of the tolerantclone. Roots of both clones contained similar amounts of copperafter removal of the readily exchangeable fraction. More copperwas translocated to the shoots of the tolerant than the non-tolerantclone. The explanation of copper tolerance in Agrostis giganteamust be sought in areas other than those of differences in grosscopper absorption and retention by roots.     

Studies on the mechanisms of barley seed deterioration

The type of chemical treatment used to control regrowth from hardwood stumps affects their colonization by fungi. Since such stumps often act as sources of infection for Armillaria mellea this may have important consequences. Ammonium sulphamate generally favours growth of species causing rapid decay, some of which compete well with A. mellea, whereas 2,4,5-trichlorophenoxyacetic acid tends to promote growth of Chondro-stereum purpureum which is replaced by A. mellea. The former treatment is recommended for situations where serious damage from A. mellea seems likely to occur, because the best hope of control is to ensure rapid killing of stump roots. Prompt colonization of stumps was obtained by inoculating the cut surface with spores of a wood-rotting fungus and then treating it with 40% ammonium sulphamate. In birch stumps jointly inoculated with Coriolus versicolor and A. mellea the amount of wood occupied by the latter after 4 yr was greatly reduced by comparison with controls inoculated with A. mellea alone. Stump inoculation might be worth developing for use in valuable plantations and other intensively managed areas such as parks and gardens, especially where measures such as stump excavation are impracticable.     

The Effect of Felling on the Occurrence of Microfungi Stimulatory to Armillaria Rhizomorph Formation in Thin Roots of Quercus robur

Eight fungal assemblages were isolated from thin branch roots (0.5–1 mm diameter) of living oak trees (Quercus robur) and their stumps 2 years after felling. The roots were serially washed or surface‐sterilized with ethanol and hypochlorite. The thin roots were inhabited by fungi that are stimulants of Armillaria rhizomorphs as found previously in thick roots (0.5–1 cm diameter). However, unlike thick roots, the densities of the fungi in serially washed roots were significantly less in stump roots than in living roots. Among the most common ‘stimulants’, the frequencies of Cylindrocarpon didymum, Mortierella gracilis, Mor. microspora var. macrocystis, Mycelium radicis atrovirens, Nectria grammicospora, Pseudogymnoascus roseus and Sporothrix schenckii were usually significantly greater in stump roots than in living roots, while the frequencies of Chrysosporium merdarium, Cy. destructans, Mor. hygrophila, Penicillium adametzii, Pe. citrinum, Pe. daleae, Pe. janczewskii and Pe. spinulosum were usually significantly less in stump roots. The frequency of Trichoderma viride was less in the stump roots. Serial washing of roots was more effective than chemical surface‐sterilization in the detection of Armillaria rhizomorph ‘stimulants’. The significance of these findings in the ecology and risk assessment of Armillaria in plantations is discussed.