Critical review of Identification Techniques for Armillaria spp. and Heterobasidion annosum Root and Butt Rot Diseases

The need for a rapid and definite diagnosis of the Armillaria spp. and Heterohasidion annosum root and butt rot diseases as well as general requirements expected of a test to detect the pathogens are reviewed. As a spin-off from more fundamental molecular biological research there has been a remarkable increase in new methods for diagnosis (i.e. detection and identification) in recent years. These new methods all have in comtnon that they use strtjctural elements of the target organisms such as proteins, polysaccharides, glycoproteins, nucleic acids, etc. for the construction of antibodies, probes and primers for detection, or to produce electrophoretic patterns to be used for classification and identification. This paper presents a short review of principles, advantages, disadvantages and some perspectives of the new methods developed for Armillaria spp. and H. annosum diagnosis compared with conventional ones, such as interfertility testing or visual inspection of the pathogens in situ and in vitro in pure culture. It will be noted that the specificity and reproducibility of the new techniques, which are closely linked with potential experimental error, lead to principal pitfalls which should be kept in mind when conclusions are drawn on data generated by them.
Even though PCR-based methods become increasingly more widespread it is likely that most diagnostic methods will continue to be used or co-exist in some form in the future.     

Development of a rapid and simple Agrobacterium tumefaciens-mediated transformation system for the fungal pathogen Heterobasidion annosum

Heterobasidion annosum causes root and butt-rot in trees and is the most serious forest pathogen in the northern hemisphere. We developed a rapid and simple Agrobacterium-mediated method of gene delivery into H. annosum to be used in functional studies of candidate genes and for visualization of mycelial interactions. Heterobasidion annosum TC 32-1 was cocultivated at pH 5.6 and 20 degrees C in Hagems medium with Agrobacterium tumefaciens C58 carrying plasmids with hygromycin B resistance as the selectable marker and green fluorescent protein as a visual marker. We obtained 18 mitotically stable transformed isolates showing green fluorescence protein activity.     

Rapid Detection of European Heterobasidion annosum Intersterility Groups and Intergroup Gene Flow Using Taxon-specific Competitive-Priming PCR (TSCP-PCR)

Using taxon-specific competitive-priming polymerase chain reaction (TSCP-PCR) the intersterility groups (ISGs) S and P of the root and butt rot pathogen Heterobasidion annosum were found in an active pine mortality center in the forest region Tharandter Wald (Saxonia, Germany). All isolates from spruce were typed as S, and all isolates from pine were P, except for two isolates which were identified as hybrid 'SP’genets. These results indicate the possibility of gene flow in nature between the two ISGs in Europe.     

Differential anatomical response of Norway spruce stem tissues to sterile and fungus infected inoculations

The anatomical defense responses in stems of Norway spruce (Picea abies) clones of different resistance to pathogenic fungi were characterized over time and distance from small mechanical wounds or wounds inoculated with the root rot fungus Heterobasidion annosum. Common responses for both treatments included division of ray parenchyma and other cells in the cambial zone, accumulation of phenolic inclusions in ray parenchyma cells, activation of phloem parenchyma (PP) cells, and formation of traumatic resin ducts (TDs) in the xylem. TD formation occurred synchronously from a tangential layer of cells, or symplasmic domain, within the zone of xylem mother cells. TD induction is triggered by a signal, which propagates a developmental wave in the axial direction at about 2.5 cm per day. TDs are formed at least 30 cm above single inoculations within 16–36 days after inoculation. The size and number of TDs is attenuated further away from the inoculation site, indicating a dose-dependent activity leading to TD development. Compared to sterile wounding, fungal inoculation gave rise to more and larger TDs in all clones, and multiple rows of TDs in weak clones. Fungal inoculation also induced the formation of more new PP cells, increasing the number of PP cells in the phloem in the year of inoculation up to 100%. TD and PP cell formation was greater in susceptible compared to resistant clones and after fungal versus sterile inoculation. Potential mechanisms responsible for this variable response are discussed.     

Evolutionary history of the conifer root rot fungus Heterobasidion annosum sensu lato

We investigated two hypotheses for the origin of the root rot fungus Heterobasidion annosum species complex: (i) that geology has been an important factor for the speciation (ii) that co-evolutionary processes with the hosts drove the divergence of the pathogen species. The H. annosum species complex consists of five species: three occur in Europe, H. annosum s.s., Heterobasidion parviporum and Heterobasidion abietinum, and two in North America, Heterobasidion irregulare and Heterobasidion occidentale; all with different but partially overlapping host preferences. The evolution of the H. annosum species complex was studied using six partially sequenced genes, between 10 and 30 individuals of each species were analysed. Neighbour-joining trees were constructed for each gene, and a Bayesian tree was built for the combined data set. In addition, haplotype networks were constructed to illustrate the species relationships. For three of the genes, H. parviporum and H. abietinum share haplotypes supporting recent divergence and/or possible gene flow. We propose that the H. annosum species complex originated in Laurasia and that the H. annosum s.s./H. irregulare and H. parviporum/H. abietinum/H. occidentale ancestral species emerged between 45 and 60 Ma in the Palaearctic, well after the radiation of the host genera. Our data imply that H. irregulare and H. occidentale were colonizing North America via different routes. In conclusion, plate tectonics are likely to have been the main factor influencing Heterobasidion speciation and biogeography.     

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.     

The importance of inoculum size for the competitive ability of wood decomposing fungi

Abstract Competition among wood decay fungi was studied with a technique using sectors of various sizes of 8 cm diameter wood discs. The sectors representing 8–92% of the discs were precolonized by Heterobasidion annosum (Fr.) Bref., Resinicium bicolor (Alb. & Schw. ex Fr.) Parm, Phanerochaete sanguinea (Fr.) Hjortstam and Coniophora sp. DC. ex Me'rat before they were combined pairwise in close contact on top of water agar in 9 cm Petri dishes. Discs were regularly inspected for mycelial overgrowth and after 10 weeks mycelia were reisolated. Competitive success, measured as the replacement of the opposing fungus, was generally greatest for mycelia with sectors representing 92% of a disc and smallest for 8% sectors. R bicolor was the most, and H. annosum the least, competitive of the species investigated. The results indicate that mycelial size could be one major factor influencing the competitive success in nature. However, when paired on nutrient agar, the results of the interactions between the four species did not correspond to those in wood discs     

Registration of Phlebiopsis gigantea as a Forest Biocontrol Agent in the UK: Recent Experience

The registration of one of the world's first biological control agents for a plant pathogen, in January 1998, after 30 years of field use, is discussed in relation to its approval under the UK Control of Pesticides Regulations 1986. The control agent, Phlebiopsis gigantea (formerly known as Peniophora gigantea), is a common wood-rotting basidiomycete which is applied to the freshly cut surfaces of pine stumps to prevent their colonization by the pathogenic rootrotting fungus Heterobasidion annosum . Unlike many other biocontrol agents, P. gigantea is not a biocide that kills the target organism. Rather, it competes for resources that the pathogen would otherwise use, providing an extension to a naturally occurring process. Experience in securing approval is reviewed, with special reference to the identity of the organism and its natural distribution, and to assessments of the risk its use might pose in the environment. It is suggested that before the formal process of applying for approval of a biocontrol agent begins, there should be procedures for agreeing precise data requirements. These should be based on the type of crop to be treated and the mode of operation and characteristics of the biocontrol organism. This would streamline the registration process.