单孢融合性实验法鉴定采自四川北部的小孔异担子菌

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。     

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.     

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.     

Species of Heterobasidion host a diverse pool of partitiviruses with global distribution and interspecies transmission

We investigated the geographic occurrence and genetic diversity of partitiviruses among 247 Heterobasidion specimens representing seven species and originating from Europe, Asia, and North America. Based on sequence analysis, partitiviruses were relatively rare, and occurred only in about 5 % of the Heterobasidion isolates analyzed, constituting a minority (about 28 %) of all virus-infected [double-stranded RNA (dsRNA)-positive] isolates. Altogether ten virus strains were characterized in sequence: one complete genome sequence of 3893 bp, six complete RNA-dependent RNA polymerase sequences of 2000-2033 bp, and three partial polymerase sequences. Based on phylogenetic analysis, the virus strains were assigned into three putative partitivirus species: HetRV1 (Heterobasidion RNA virus 1), HetRV4, and HetRV5. Degenerate consensus primers were designed for RT-PCR detection of these virus species. HetRV1 occurred in five different Heterobasidion species, and resembled the previously described Heterobasidion annosum virus (HaV). Highly similar HetRV1 strains with 98 % nucleotide level similarity were found from H. parviporum (member of the H. annosum species complex) and H. australe (member of the H. insulare complex) growing in the same region in Bhutan. This observation suggests recent virus transmission between these taxonomically distant Heterobasidion species in nature. It was also shown that HetRV1 can be transmitted by mycelial contact between the H. annosum and H. insulare complexes. The two other virus species, HetRV4 and HetRV5, were closely related to the Amasya Cherry Disease-associated mycovirus, to Heterobasidion parviporum partitivirus Fr110B, and also to several plant-infecting alphacryptoviruses. These results are in accordance with the view of a close evolutionary relationship between partitiviruses of plants and fungi.     

Comparison of Three Products Based on Phlebiopsis gigantea for the Control of Heterobasidion annosum in Europe

The fungus Phlebiopsis gigantea has been used in Europe as a biological agent for the control of conifer root and butt (caused by Heterobasidion annosum ) for nearly 40 years. P. gigantea competes with H. annosum for the woody resource within conifer stumps, and is applied to stump surfaces at felling. Three distinct biological control products based on P. gigantea have been developed: PG Suspension in the UK, PG IBL in Poland and Rotstop in Finland. The formulations are of oidia, which are maintained in a sucrose suspension, sawdust, or a wettable powder, respectively. PG Suspension and PG IBL are applied to pine stumps, while Rotstop is equally as effective on pine as on Norway spruce stumps. For each product, isolates of P. gigantea are selected from the wild and are screened for their competitive ability against H. annosum before formulation. Viability and purity checks are undertaken throughout the production cycle and during routine use. The increasing use of mechanized harvesting machines to fell and process trees is having an impact on this biological control system, the formulations having to be compatible with the mechanical application systems and vice versa. This paper compares the formulation, testing and application of the three products, and considers some aspects of their future development.     

Iron and reactive oxygen responses in Pinus sylvestris root cortical cells infected with different species of Heterobasidion annosum sensu lato

Defence mechanisms in trees are not well understood. We assessed whether distribution of iron ions and their co-localisation with reactive oxygen species in Pinus sylvestris root cells reflect differential preferences of the pathogens Heterobasidion annosum sensu stricto, H. parviporum and H. abietinum to the host. Strains of H. annosum s.s. characterised by a greater preference for P. sylvestris induced accumulation of superoxide (O(2) (-)) in host cells 6?h after inoculation, whereas two peaks in accumulation of O(2) (-) (after 4 and 48?h) were observed after infection with strains of the pathogens H. parviporum and H. abietinum, which have a lower preference for P. sylvestris. Moreover, strains of H. annosum s.s. caused increased production of hydrogen peroxide (H(2)O(2)) in P. sylvestris cells, in contrast with strains of the other two species (H. parviporum and H. abietinum). Following inoculation with H. annosum s.s. strains, H(2)O(2) was correlated negatively with O(2) (-) and correlated positively with ferrous iron (Fe(2+)). Co-localisation of Fe(3+) with H(2)O(2) may suggest that they are involved in inducing hypersensitive responses and eventually cell death in roots inoculated with H. annosum s.s. strains, in contrast with H. parviporum, in which other mechanisms operate when the host is parasitised.     

Heterologous array analysis in Heterobasidion: hybridisation of cDNA arrays with probe from mycelium of S, P or F-types

Because of the close relatedness between three species of Heterobasidion annosum (P-type), Heterobasidion parviporum (S-type) and Heterobasidion abietinum (F-type), we investigated the possible use of arrays from one species for studies of gene expression in the other. Clones containing partial cDNAs from 94 identifiable genes expressed during spore germination and differentiation in H. parviporum were printed manually in six replications on nylon membranes. The membrane was hybridized with chemifluorescent labelled cDNA from actively growing mycelia of H. parviporum, H. annosum or H. abietinum, cultivated on a non-selective substrate. Product-moment correlation coefficient varied between 0.81 and 0.49. Due to the level of correlation, in the gene expression among the intersterility groups, we concluded that the cDNA array of one can be used to study gene expression in the others.     

Modelling the mechanisms behind the key epidemiological processes of the conifer pathogen Heterobasidion annosum

The Heterobasidion annosum species complex is a widely distributed group of fungal conifer pathogens causing root and butt rots. We studied the key processes of Heterobasidion epidemiology by compiling models that rely on biological processes. Models were included in the mechanistic model with stochastic elements, Hmodel, simulating the fungal dynamics in even-aged Norway spruce stands. The results from the modelling and stand-level simulations indicated that primary infections are affected by the stump size and spore deposition. In addition, we found that Heterobasidion dynamics at the scale of the stand are driven by several infections in large stumps, rather than by numerous infections in small stumps. We assessed the need for quantitative results in Heterobasidion biology, especially in the spread mechanisms, to support the development of complex mechanistic models.     

Spruce wood degradation by Pleurotus abieticola in comparison with Phlebiopsis gigantea and Heterobasidion parviporum: in vitro experiments with isolates

The objective of this study was to evaluate the in vitro mycelium growth of Pleurotus abieticola and its competitive ability to decompose sapwood and heartwood wood, as compared to the activity of Phlebiopsis gigantea and Heterobasidion parviporum. Over the last several decades, P. gigantea has routinely been used for biocontrol of the conifer pathogen Heterobasidion annosum s.l.; however, its protective effect on Norway spruce stands was recently demonstrated to be not satisfactory. P. abieticola was proposed instead, as a promising species that might successfully compete with H. parviporum. We investigated the growth of mycelium and the ability of P. abieticola isolates to decompose wood of Norway spruce, in the experiment with isolates of P. gigantea and H. parviporum. Heartwood was better decomposed than sapwood by the majority isolates used in the experiment. Linear growth of the investigated fungi showed a more rapid mycelium development for P. gigantea and H. parviporum, compared to that of P. abieticola. In dual cultures, H. parviporum was overgrown only by P. gigantea. All the tested isolates of P. abieticola showed weaker wood decomposition than those of P. gigantea and H. parviporum. Further study is required to better understand the role of P. abieticola for the protection of spruce stands.     

Intraspecific variation in Heterobasidion annosum for mortality rate on Pinus sylvestris and Picea abies seedlings grown in pure culture

One-month-old Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings were inoculated in vitro with Heterobasidion annosum strains, four each of the P-, S- and F-intersterility groups. Variation among strains and between the IS groups in virulence, expressed in mortality rate, was detected during twelve months after inoculation. Most of the strains were more virulent on spruce than on pine, and mortality of spruce seedlings was significantly higher. The P strains displayed similar virulence on both hosts, while S strains caused higher mortality of spruce seedlings and significantly lower mortality of pine seedlings. Strains of the F group were less virulent, but killed significantly (P < 0.05) more spruce than pine seedlings. In the interspecific analyses with two hosts, the isolates and IS groups accounted for most of the explained variation in the host mortality.     

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.     

Genetic Relatedness Among Strains of Heterobasidion annosum as Detected by Random Amplified Polymorphic DNA Markers

Heterobasidion annosum, the casual agent of root and butt rots of trees is one of the most important plant pathogens in temperate coniferous regions of the world. It is comprised of several intersterility groups with different host preferences. Of the H. annosum isolates from various geographical regions belonging to various intersterility groups, 36 showed group-specific banding patterns in RAPD assay. More polymorphisms were found within the S- and F-group isolates than in the P group. Genetic similarities among the isolates were determined by UPGMA clustering revealing the presence of four major clusters: P, S, F, and the North American S. The P group was the most distinctive of all the other groups, while the European S and F and the North American S were more closely related. The P group showed only 20% similarity to the other groups and may be referred to as a subspecies. The North American S group was separate from the European S group. The potential influence of the historical spread of coniferous trees in the isolate genetic differentiation in Europe is discussed.     

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.     

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.     

Co-inoculation of Ceratocystis polonica and Heterobasidion annosum with callus of two Norway spruce clones with different in vivo susceptibility

We have studied how callus cultures from two clones of Norway spruce influence the growth of two pathogens, Ceratocystis polonica and Heterobasidion annosum, when co-cultivated in vitro. In field experiments, trees of clone 409 were susceptible to both fungi, whereas clone 589 was less affected. Callus was cultured on medium containing cytokinins (benzylaminopurine, kinetin) and with or without auxin (2,4-dichlorophenoxyacetic acid). For co-cultivation with fungus, one piece of callus was placed towards the edge of each Petri dish. One and 14 days after inoculation with callus the dishes were co-inoculated with the fungus. Both clones strongly stimulated the initial growth of both fungi. Clone 589 inhibited the growth of both fungi when the fungi were inoculated one day after the callus. When the callus was cultured on medium without auxin for 14 days before co-inoculation clone 589 strongly inhibited the growth of both fungi, whereas clone 409 inhibited H. annosum only. This revised version was published online in June 2006 with corrections to the Cover Date.     

Long-term effects on other fungi are studied in biological and chemical stump treatments in the fight against Heterobasidion annosum coll

The effects on mycoflora of Norway spruce stumps of biological and chemical treatments in the fight against Heterobasidion annosum coll. were investigated two yr after applications of these treatments. The biological treatments were Hypholoma fasciculare, Phanerochaete velutina, Vuilleminia comedens, Trichoderma harzianum and both the conidial suspension and culture filtrate of Verticillium bulbillosum; propiconazole was used as chemical treatment. Samplings were performed on 130 stumps, including controls with (C1) and without (C2) an autologous wood disk. Forty-nine fungal taxa were isolated, and most were Deuteromycetes. Trichoderma harzianum significantly reduced the number of taxa versus controls (three versus 25), while the other treatments showed more limited qualitative and quantitative effects. Cluster and correspondence analysis differentiated three groups of treatments: one including the three Basidiomycetes, V. bulbillosum and C1; one comprising propiconazole and C2; and one composed of the treatment with T. harzianum only. Because the same stumps already had been sampled one yr after treatments in a similar study, comparisons between data were possible and were very useful in the investigation of the temporal evolution of the effects of each treatment. Multivariate analysis showed that the strong effects of T. harzianum on stump mycocenoses increased over time. Transient effects were shown in most treatments (i.e., the three Basidiomycetes), whereas V. bulbillosum had the least impact on naturally occurring mycoflora.