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.     

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.     

Microsatellite markers for the diploid basidiomycete fungus Armillaria mellea

We isolated and characterized 12 microsatellite markers for two North American populations (California, Pennsylvania) of Armillaria mellea, a fungal pathogen responsible for Armillaria root disease of numerous woody plants. Allele frequency ranged from two to nine alleles per locus, and gene diversity ranged from 0.05 to 0.86. Of the 12 loci, eight loci were polymorphic in the California and Pennsylvania populations, and showed no evidence of heterozygote deficiencies or severe linkage disequilibrium. Our results suggest that we have isolated and characterized variable loci to estimate genotypic diversity, gene flow and migration, and to determine population structure of North American A. mellea.     

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。     

High phenotypic variability in the wood decay fungus Phellopilus nigrolimitatus

Genetic and phenotypic variation among individuals increases population resilience to external disturbances and enables quicker adaptation to changing environments. Since the predominant phase in the fungal lifecycle occurs belowground or within substrates, it is difficult to assess the level of variation in functional traits occurring within species in natural environments. In this study, we investigated phenotypic variation in the wood-decay fungus Phellopilus nigrolimitatus and related this to genet size, measured as vegetative compatibility groups (VCGs). Out of 321 wood samples and sporocarps collected on six logs of Picea abies, we isolated 230 dikaryotic isolates that were grouped into 53 VCGs based on culturing experiments. The number of VCGs per log varied from six to twelve, with large variation in sizes; the smallest VCG was derived from a single wood sample, while the largest was isolated across 31 wood samples stretching over 15 m. The size of the VCGs was positively correlated with the area of the spore-producing layer of the sporocarps, implying that larger genets produce more spores. Additionally, the phenotypic variation among isolates of P. nigrolimitatus was high, both in terms of growth morphology and coloration, and consistent with the delimitation of isolates into VCGs. Isolates could to a large extent be correctly assigned to the VCGs a priori based purely on culture phenotype. This study reveals that extensive phenotypic variation can be observed between fungal genets at very fine spatial scale, and calls for more studies looking at intraspecific phenotypic variation, in line with assessing genetic diversity in fungi.     

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.     

Microsatellite markers for population studies of the rice blast fungus,Magnaporthe grisea

We developed nine new microsatellite markers for rice blast (Magnaporthe grisea) population studies. These markers were used in addition to nine microsatellite markers previously developed by our group for mapping purpose. Altogether, the 18 markers were used in multiplex PCR (polymerase chain reaction) to characterize six populations from different geographical origins. The average number of alleles per locus across populations ranged from 1.2 to 7 and the total number of alleles detected from 2 to 19. Based on this large range of polymorphism, this set of markers is expected to be useful for different kind of population studies at different geographical scales.     

Biological Processing of Pine Logs for Pulp and Paper Production with Phlebiopsis gigantea

Phlebiopsis gigantea (=Phanerochaete gigantea) is a white rot fungus that rapidly colonizes cut stumps, stems, and branches of pine. Two laboratory and several field studies showed that inoculation of red pine logs, Pinus resinosa, with P. gigantea reduced the pitch content of wood, facilitated bark removal, modified wood cells, and controlled detrimental sapstain. Isolations from inoculated logs revealed up to 100 and 80% colonization of the sapwood by P. gigantea after 8 weeks in the field and 32 days in the laboratory, respectively. Logs colonized by P. gigantea in both the laboratory and field showed a 9 to 71% reduction in pitch content, as well as a significant enhancement of bark removal. Examination with Simons' stain of refined wood fibers from inoculated logs revealed an increase in cell wall porosity. Blue stain fungi that cause dark discoloration of the sapwood were inhibited by inoculation with P. gigantea. These studies demonstrate that biological processing of logs with P. gigantea can result in substantial benefits to the pulp and papermaking process.     

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.     

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.     

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.     

Phlebiopsis gigantea and associated viruses survive passing through the digestive tract of Hylobius abietis

Phlebiopsis gigantea (Fr.) Jül. is one of the most common fungal species in coniferous forests and commonly used as a biological control agent to prevent aerial infections of conifers by Heterobasidion fungi. We used feeding experiments to examine whether Hylobius abietis L. (Coleoptera: Curculionidae) could serve as a vector for P. gigantea and associated viruses by disseminating the fungus in its faecal pellets. Two different P. gigantea strains were used in the experiments: the virus-free Rotsop biocontrol strain and P. gigantea 93073 infected with the virus strain PgLV-1. The Rotstop strain showed 100% viability during insect feeding, while the viability of the virus-infected strain 93073 was only 35%. Virus persistence was 100% during the passage of the host fungus through the alimentary tract. Based on growth experiments using virus-infected and virus-free strains obtained by hyphal tip or oidial isolation, the presence of PgLV-1 did not significantly affect the growth rate of the host fungus.     

Microsatellite markers for Hebeloma species developed from expressed sequence tags in the ectomycorrhizal fungus Hebeloma cylindrosporum

The increasing number of expressed sequence tag (EST) projects dedicated to ectomycorrhizal fungi is translating into the release of large sets of ESTs. The aim of this study was to develop and test simple sequence repeat (SSR) markers from EST databases of the model ectomycorrhizal fungus Hebeloma cylindrosporum. Six SSR markers were found to be both unambiguously scorable and polymorphic among 12 H. cylindrosporum isolates. Two SSR markers were transferable to other Hebeloma species and one marker was interestingly found to be polymorphic among seven H. crustuliniforme isolates.     

Beetles provide directed dispersal of viable spores of a keystone wood decay fungus

Wood decay fungi are considered to be dispersed by wind, but dispersal by animals may also be important, and more so in managed forests where dead wood is scarce. We investigated whether beetles could disperse spores of the keystone species Fomitopsis pinicola. Beetles were collected on sporocarps and newly felled spruce logs, a favourable habitat for spore deposition. Viable spores (and successful germination) of F. pinicola were detected by dikaryotization of monokaryotic bait mycelium from beetle samples. Viable spores were on the exoskeleton and in the faeces of all beetles collected from sporulating sporocarps. On fresh spruce logs, nine beetle species transported viable spores, of which several bore into the bark. Our results demonstrate that beetles can provide directed dispersal of wood decay fungi. Potentially, it could contribute to a higher persistence of some species in fragmented forests where spore deposition by wind on dead wood is less likely.     

Microsatellite markers for European Daphnia

We present 32 polymorphic microsatellite markers for species of the European Daphnia longispina group: D. galeata, D. hyalina, D. rosea, D. cucullata and D. curvirostris. Microsatellite markers were either isolated from genomic libraries or optimized based on previously published sequence information of sister taxa. Cross‐species tests revealed that all but one of the polymorphic markers are applicable to more than one species, which allows intra‐ and interspecific genetic studies on, i.e. population structure, hybridization events and introgression.     

Microsatellite markers for the whelk Buccinum undatum

Five microsatellite loci are described for the commercially exploited marine gastropod, Buccinum undatum. Levels of polymorphism were variable with three to 19 alleles per locus and expected heterozygosities of 0.26–0.94 in 60 individuals of the population from which the loci were isolated. Homozygote excess at two of the loci might be attributable to null alleles, and these loci should not be used in, for example, parentage analysis. Nevertheless, because null allele frequencies can be estimated and their effects partitioned, all are useful markers for studies of population differentiation.     

Microsatellite markers for the earthworm Lumbricus rubellus

We describe eight microsatellite markers for the earthworm Lumbricus rubellus, a commonly used ‘biological sentinel’ species in ecotoxicology studies. Thirty‐four individuals from a single population were tested for polymorphism. Markers possessed between two and 15 alleles per locus, and expected heterozygosity ranged between 0.06 and 0.92 (observed heterozygosity 0.03–0.92). Unfortunately, no consistent cross‐species amplification was observed in the related congeners Lumbricus castaneus and Lumbricus terrestris.