Antrodia gossypium,Phlebiopsis gigantea and Heterobasidion parviporum: in vitro growth and Norway spruce wood block decay

Polymerase chain reaction-amplified and sequenced isolates of Antrodia gossypium, Phlebiopsis gigantea and Heterobasidion parviporum from decaying Norway spruce wood blocks after three and six months, which exhibited linear growth, were investigated. P. gigantea strains showed the fastest growth, whereas A. gossypium growth was five times slower. The differences between the mean daily increment of A. gossypium and the other examined isolates (except Hp2) were statistically significant. There were also significant differences in wood decay between densities over time. These results were confirmed by the decay acceleration index (DAI) and decay activity index, which were positively correlated with wood density regardless of the fungus species. The registered P. gigantea strains (Rotstop and PG Suspension) exhibited a strong decomposition ability (28% after six months); the weight loss caused by A. gossypium after six months of decay (15.2%) was similar to the results of P. gigantea (GB) after just three months (13.2%). All tested H. parviporum isolates showed rather rapid growth and equally strong wood decay (20–25%) compared to those of P. gigantea. DAI showed that A. gossypium may significantly contribute to wood decomposition over time, particularly in less dense wood samples. The use of both saprotrophs as biological agents against root pathogens is discussed.     

The primary module in Norway spruce defence signalling against H. annosum s.l. seems to be jasmonate-mediated signalling without antagonism of salicylate-mediated signalling

A key tree species for the forest industry in Europe is Norway spruce [Picea abies (L.) Karst.]. One of its major diseases is stem and butt rot caused by Heterobasidion parviporum (Fr.) Niemelä & Korhonen, which causes extensive revenue losses every year. In this study, we investigated the parallel induction of Norway spruce genes presumably associated with salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways previously observed in response to H. parviporum. Relative gene expression levels in bark samples of genes involved in the salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways after wounding and inoculation with either the saprotrophic biocontrol fungus Phlebiopsis gigantea or with H. parviporum were analysed with quantitative PCR at the site of the wound and at two distal locations from the wound/inoculation site to evaluate their roles in the induced defence response to H. parviporum in Norway spruce. Treatment of Norway spruce seedlings with methylsalicylate, methyljasmonate and inhibitors of the jasmonic acid/ethylene signalling pathway, as well as the Phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid were conducted to determine the responsiveness of genes characteristic of the different pathways to different hormonal stimuli. The data suggest that jasmonic acid-mediated signalling plays a central role in the induction of the genes analysed in this study irrespective of their responsiveness to salicylic acid. This may suggest that jasmonic acid-mediated signalling is the prioritized module in the Norway spruce defence signalling network against H. parviporum and that there seems to be no immediate antagonism between the modules in this interaction.     

Association genetics identifies a specifically regulated Norway spruce laccase gene,PaLAC5, linked to Heterobasidion parviporum resistance

It is important to improve the understanding of the interactions between the trees and pathogens and integrate this knowledge about disease resistance into tree breeding programs. The conifer Norway spruce (Picea abies) is an important species for the forest industry in Europe. Its major pathogen is Heterobasidion parviporum, causing stem and root rot. In this study, we identified 11 Norway spruce QTLs (Quantitative trait loci) that correlate with variation in resistance to H. parviporum in a population of 466 trees by association genetics. Individual QTLs explained between 2.1 and 5.2% of the phenotypic variance. The expression of candidate genes associated with the QTLs was analysed in silico and in response to H. parviporum hypothesizing that (a) candidate genes linked to control of fungal sapwood growth are more commonly expressed in sapwood, and; (b) candidate genes associated with induced defences are respond to H. parviporum inoculation. The Norway spruce laccase PaLAC5 associated with control of lesion length development is likely to be involved in the induced defences. Expression analyses showed that PaLAC5 responds specifically and strongly in close proximity to the H. parviporum inoculation. Thus, PaLAC5 may be associated with the lignosuberized boundary zone formation in bark adjacent to the inoculation site.     

Spread of Heterobasidion annosum s.s. and Heterobasidion parviporum in Picea abies 15 years after stump inoculation

The tree pathogenic fungi Heterobasidion annosum s.s. and Heterobasidion parviporum cause root and butt rot in Norway spruce (Picea abies) and produce serious economic losses to the forest sector in Europe. We experimentally studied inter- and intraspecific differences between H. parviporum and H. annosum s.s. in the way they infect stumps and spread into neighbouring trees. Eleven H. parviporum and nine H. annosum s.s. isolates were artificially inoculated on stumps of two spruce stands after first thinning. After 15 years, the same isolates were reisolated from neighbouring trees. Heterobasidion parviporum spread more frequently from the inoculated stumps to the neighbouring trees than H. annosum s.s. The surroundings of H. annosum s.s. stumps that did not spread were often colonized by H. parviporum. Heterobasidion annosum s.s. spread was restricted mainly to the areas of the plot where no other Heterobasidion genotypes had been inoculated. In such cases, H. annosum s.s. tended to develop into bigger genets than H. parviporum. The probability of stump-to-tree spread of H. parviporum depended on the diameter of the stumps, suggesting that H. parviporum spread may relate to the presence of heartwood. Both H. parviporum and H. annosum s.s. proved to be strong pathogens on Norway spruce; however, when competing for the same trees, H. parviporum seemed capable of excluding H. annosum s.s. from the stand.     

Characterisation of some Phlebiopsis gigantea isolates with respect to enzymatic activity and decay of Norway spruce wood

The activity of cellulase, peroxidase, phosphatase and dehydrogenase enzymes, together with the content of protocatechuic and vanillic acids, in samples of Norway spruce wood inoculated with 17 different isolates of Phlebiopsis gigantea was measured. The same isolates were used to compare decay activity in samples of Norway spruce wood after incubation for 3 and 6 months. Significant differences in enzyme activity and phenol production were found between aerial mycelium overgrowing the wood sample and the underlying wood. These differences indicated that the nature of the fungal mycelium appears to change depending on whether it is in contact with wood. After 6 months, highly extensive decomposition of the wood was shown by two British isolates. The results confirm a large difference in P. gigantea inoculum among isolates in natural conditions and reinforce the need for constant evaluation of the most active isolates to use in preparations for biocontrol: a problem for both users and registration bodies.     

Spatial Patterns in Hyphal Growth and Substrate Exploitation within Norway Spruce Stems Colonized by the Pathogenic White-Rot Fungus Heterobasidion parviporum

In Norway spruce, a fungistatic reaction zone with a high pH and enrichment of phenolics is formed in the sapwood facing heartwood colonized by the white-rot fungus Heterobasidion parviporum. Fungal penetration of the reaction zone eventually results in expansion of this xylem defense. To obtain information about mechanisms operating upon heartwood and reaction zone colonization by the pathogen, hyphal growth and wood degradation were investigated using real-time PCR, microscopy, and comparative wood density analysis of naturally colonized trees with extensive stem decay. The hyphae associated with delignified wood at stump level were devoid of any extracellular matrix, whereas incipient decay at the top of decay columns was characterized by a carbohydrate-rich hyphal sheath attaching hyphae to tracheid walls. The amount of pathogen DNA peaked in aniline wood, a narrow darkened tissue at the colony border apparently representing a compromised region of the reaction zone. Vigorous production of pathogen conidiophores occurred in this region. Colonization of aniline wood was characterized by hyphal growth within polyphenolic lumen deposits in tracheids and rays, and the hyphae were fully encased in a carbohydrate-rich extracellular matrix. Together, these data indicate that the interaction of the fungus with the reaction zone involves a local concentration of fungal biomass that forms an efficient translocation channel for nutrients. Finally, the enhanced production of the hyphal sheath may be instrumental in lateral expansion of the decay column beyond the reaction zone boundary.To grow to great heights, trees continually replace their water- and nutrient-conducting elements. Older elements, such as the heartwood that is formed in many trees, gradually become nonconductive. In contrast to the living sapwood, heartwood lacks active defense mechanisms against microbes. However, lignin, the polymer coating cell wall polysaccharides, is highly resistant to microbial degradation. In fact, white-rot fungi, besides having evolved the ability to tolerate or detoxify the secondary metabolites accumulating in heartwood, are the only organisms capable of efficiently degrading lignin. Following establishment in the heartwood of living trees, the colonies of pathogenic white-rot fungi expand and eventually also threaten the conductive sapwood.The white-rot fungus Heterobasidion annosum sensu lato, composed of three species with overlapping geographic distributions and host ranges in Europe (23), is the most important pathogen of Norway spruce (Picea abies L. Karst) in boreal forests. Primary infection of Norway spruce stands by H. annosum sensu lato takes place through fresh thinning stumps or wounds on roots and at the base of the stem. Basidiospores landing on these entrance points give rise to mycelia which colonize the root systems, and eventually the fungus spreads into the stem heartwood. At sites infested with Heterobasidion parviporum, a species primarily restricted to Norway spruce, roots of saplings can become infected by the fungus after around 10 years of growth (25). Stem colonization usually initiates only after the heartwood has started to develop, which in Norway spruce takes place in trees 25 to 40 years old (17). Due to relatively rapid axial spread within heartwood, the decay column caused by H. annosum sensu lato often is up to 10 m high in the stems of mature Norway spruce trees.In response to sapwood challenge by an expanding heartwood-based colony of H. annosum sensu lato, Norway spruce forms a so-called reaction zone (RZ) in the border area between healthy sapwood and colonized heartwood. This xylem defense is characterized by high pH due to increased carbonate content and enrichment of phenolic compounds, particularly lignans, some of which have shown antifungal properties in bioassays (14, 30, 31). Although several wood decay fungi are able to eventually penetrate the RZ regions formed in trees, the strategies employed by fungi to breach these unique defense barriers are poorly understood (24). The purpose of this study was to obtain information about the mechanisms operating in heartwood colonization and expansion of the decay column via penetration of the RZ. To do this, we examined spatial growth of H. parviporum and the associated substrate exploitation patterns within naturally colonized mature stems of Norway spruce.     

Decay indices for evaluating wood decomposition activity

The goal of the current study was to evaluate the efficiency of Norway spruce wood decomposition by the saprotrophic fungus Phlebiopsis gigantea (Fr.) Jülich. We identified the following indices as reliable measures of wood decay: decay acceleration index (DAI), final decay index (FD), and decay intensity index (DI). DAI was used to assess acceleration and deceleration of spruce wood loss, FD to estimate the final wood decay, and DI to evaluate the ability of the fungus to decompose wood, taking into consideration changes in mycelial activity over time. We tested the hypothesis that the wood decay activity of any fungus can be evaluated more objectively when: (i) several isolates of the tested fungus are assessed simultaneously, (ii) tests are performed for at least two time-points, and (iii) samples vary in wood density. Allowing a three-month period for wood decomposition was inadequate for reliable assessment of the wood decay ability of P. gigantea. Conversely, six months after inoculation, the tested isolates showed significant differences in their ability to generate dry wood loss, which depended on wood density. In view of these results, the DAI, FD, and DI indices are practical tools for assessing fungal activity to predict wood loss.     

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.     

Virus Community Dynamics in the Conifer Pathogenic Fungus Heterobasidion parviporum Following an Artificial Introduction of a Partitivirus

Viruses infecting the conifer pathogenic fungus Heterobasidion annosum sensu lato are intracellular and spread via anastomosis contacts. In the laboratory, these viruses transmit readily even between somatically incompatible isolates, but their dispersal capacity in natural conditions has not been previously studied. We introduced a mycovirus to a heavily diseased forest site by inoculating Norway spruce stumps with heartrot decay using a mycelial suspension of Heterobasidion parviporum strain RT3.49C hosting the partitivirus strain HetRV4-pa1. The Heterobasidion population at the sample plot was screened for mycoviruses prior to and after the inoculation. Based on sequence analysis, the resident H. parviporum strains harbored six different strains of the virus species Heterobasidion RNA virus 6 (HetRV6) and one strain of HetRV4 prior to the inoculation. After three growth seasons, the inoculated H. parviporum host strain was not detected, but the introduced virus had infected two resident H. parviporum genets. The presence of a preexisting HetRV6 infection did not hinder spread of the introduced partitivirus but resulted in coinfections instead. The resident HetRV6 virus population seemed to be highly stable during the incubation period, while the single indigenous HetRV4 infection was not detected after the inoculation. In laboratory infection experiments, the introduced virus could be transmitted successfully into all of the resident H. parviporum genets. This study shows for the first time transmission of a Heterobasidion virus between somatically incompatible hosts in natural conditions.     

Use of a breeding approach for improving biocontrol efficacy of Phlebiopsis gigantea strains against Heterobasidion infection of Norway spruce stumps

Sixty-four wild heterokaryotic isolates of Phlebiopsis gigantea were analysed for asexual spore production, growth rate and competitive ability against Heterobasidion in vitro , as well as growth rate in Norway spruce wood. These P. gigantea traits were considered important for controlling infection of Norway spruce stumps by spores of Heterobasidion spp. Ten most promising P. gigantea isolates were crossed with each other and 172 F₁ progeny heterokaryons were analysed for the above-mentioned traits. Thirteen most promising progeny heterokaryons were selected and their biocontrol ability against infection by Heterobasidion was compared with the parental isolates in stem pieces of Norway spruce. The results indicated that the progeny strains had generally better traits and control efficacy than the parental strains. The genetic effects accounted for a part of the variations between progeny and parental strains. This further suggests that there is a potential to improve the biocontrol properties of P. gigantea through breeding.     

Does temperature regime govern the establishment of <Emphasis Type="Italic">Heterobasidion annosum</Emphasis> in Scandinavia?

We explored the reasons underlying the biogeographic distribution patterns of the economically important, wood-rotting basidiomycete Heterobasidion annosum in Sweden. Despite the commonness of suitable host trees, Heterobasidion annosum has not been recorded in the north of Sweden, whereas its relative, H. parviporum, is present throughout the country. To test the hypothesis that H. annosum has not spread to the north because of the effect of climate, mainly differences in the general temperature regime, we inoculated Norway spruce stumps and standing trees with H. annosum and H. parviporum at six field sites, three in the south and three in the north of Sweden. Three strains of both species were used in random combinations, so that each selected stump and tree was inoculated with both species at the same time. At 2 and 10 months after the inoculations, we compared the frequencies of detection of H. annosum and H. parviporum colonies at different distances from inoculation points in the stumps and in trees. The H. annosum colonies were detected only infrequently on disks cut from the inoculated stumps (0–4% of re-isolations) in both areas, whereas H. parviporum was detected much more frequently (26–47% of re-isolations). In standing trees, colonies belonging to H. annosum could be detected up to 210 cm (south) and 80 cm (north) and those belonging to H. parviporum up to 210 cm (south) and 140 cm (north) above the inoculation points. Our results suggest that difference in temperature regime does not provide an explanation for the distribution limit of H. annosum.     

A peroxidase gene family and gene trees in Heterobasidion and related genera

Four putative peroxidase-encoding gene fragments, named mnp1a, mnp1b, mnp2 and mnp3, were amplified with degenerative primers from the white-rot basidiomycete genus Heterobasidion. The fragments were cloned and sequenced. Similar fragments were produced and analyzed from the related genera Amylostereum, Bondarzewia and Echinodontium. Each amplified fragment contains three identically positioned introns. According to the predicted amino acid sequence, these fragments are most similar to two Mn peroxidase-encoding genes (MPGI and mnp2) and gene pgv of Trametes versicolor. Conserved residues thought to be essential for peroxidase function were identified. All four MnP gene loci of Heterobasidion were detected only in H. parviporum. Variation occurred in the predicted amino-acid sequences (131-132 amino acids) of all four fragments originating from the 47 Heterobasidion isolates tested. Amino acid variation in fragments of mnp2 and mnp3 separated European Heterobasidion parviporum ("S-type") and H. abietinum ("F-type"), known to have identical rDNA sequences. Asian and western North American isolates from fir, spruce and other hosts had the peroxidase amino acid sequences of European H. parviporum. American and European H. annosum ("P-type") isolates had different amino acid sequences and might be cryptic species.     

Suppression of plant defence response by a mycorrhiza helper bacterium

The aim of the present study was to determine whether the mycorrhiza helper bacterium Streptomyces sp. AcH 505 could serve as a biocontrol agent against Heterobasidion root and butt rot. Bacterial influence on mycelial growth of Heterobasidion sp. isolates, on the colonization of wood discs and Norway spruce (Picea abies) roots was determined. The effect of AcH 505 on plant photosynthesis, peroxidase activity and gene expression, and needle infections were investigated. AcH 505 was antagonistic to 11 of 12 tested fungal Heterobasidion isolates. The antagonism resulted in a suppression of fungal colonization of Norway spruce roots and wood discs. Mycelial growth rate of the 12th strain, Heterobasidion abietinum 331 was not affected by AcH 505, and colonization of roots by this fungal strain was promoted by AcH 505. Bacterial inoculation led to decreased peroxidase activities and gene expression levels in roots. AcH 505 promotes plant root colonization by Heterobasidion strains that are tolerant to antifungal metabolites produced by the bacterium. This may result from unknown bacterial factors that suppress the plant defence response.     

The elemental content in the mycelium of the ectomycorrhizal fungus Piloderma sp. during the colonization of hardened wood ash

Piloderma sp., a wood ash-colonizing ectomycorrhizal (EM) fungus, was grown symbiotically with Norway spruce in microcosms which contained granules of hardened wood ash. Mycelium close to the granules was sampled 3 times over a period of 11 weeks and the elemental content was investigated with particle induced X-ray emission. Mycelium from microcosms without wood ash was used as controls. The contents of P and K were similar in mycelium growing close to wood ash granules to those in control mycelium, while the Ca content increased from 23±21 mg g^–1 in controls to 63±8 mg g^–1 in mycelium growing close to wood ash granules. The Ca content was also increased in other parts of the mycelium more distant from the wood ash. Piloderma sp. may have a role in the short-term storage of Ca released from wood ash, rather than in releasing and storing P.     

大伏革菌防治小孔异担子菌的菌株筛选

以从芬兰引进的野生大伏革菌、中国的大伏革菌和小孔异担子菌为研究对象,通过平板对峙培养方法,从16株大伏革菌菌株中筛选出3株防治异担子菌的高效菌株,分别为:04052、08076和08077。这3号菌株具有较高的生长速率和拮抗率,能够快速覆盖病原菌,起到显著的生防效果。实验中还发现大伏革菌生长速率和拮抗率之间存在着显著的正相关关系。     

单孢融合性实验法鉴定采自四川北部的小孔异担子菌(英文)

在四川北部九寨沟和黄龙保护区的云杉、松树和铁杉上发现异担子菌 ,并从 6号标本中分离到 73个单孢菌株。在每个标本中随机选取 2个菌株分别与欧洲的原始多年异担子菌、小孔异担子菌和冷杉异担子菌的单孢菌株进行融合性交配。试验表明 ,这 6号标本都是小孔异担子菌。四川的菌株与欧洲的原始多年异担子菌交配不融合 ,而与欧洲的小孔异担子菌完全融合 ,并在交配后的菌落中形成锁状联合 ,且在交配的菌落中不产生拮抗线。虽然四川的菌株与欧洲的冷杉异担子菌有较高的融合性 ,但这些交配大部分为单项交配 ,即只在四川一侧的菌落中产生锁状联合 ,而且在交配的菌落中多数产生拮抗线。研究样品全部采自天然林 ,小孔异担子菌在四川经营林分中的致病性还有待进一步调查。