Fungal Diversity in Multiple Post-harvest Aged Red Pine Stumps and Their Potential Influence on Heterobasidion Root Rot in Managed Stands Across Minnesota

Thinning operations that occur in managed red pine (Pinus resinosa) stands, create tree stumps that can serve as a habitat for fungi, especially Heterobasidion irregulare, the cause of a serious root disease. Different fungi can colonize stumps early and the community of fungi can change over time as initial fungal species become replaced. Samples were collected from both the native and non-native range of red pine from stumps that were cut at different time periods. Stumps that were harvested at 0–1, 2–3, 5–6, and 10–12 years before sampling were used to provide data on the diversity of fungi that colonize tree stumps and how these communities can change over time as well as how they influence colonization of H. irregulare. Traditional culturing methods and Illumina MiSeq sequencing were used to identify the fungi in the samples. Of particular interest was Phlebiopsis gigantea, which can colonize cut stumps and prevent H. irregulare from becoming established. Overall, P. gigantea was the most abundant fungus isolated and sequenced via Illumina MiSeq. Results show that Phlebiopsis gigantea was isolated from 90% of all stumps sampled for sites harvested within 3 years of sampling in the native range of red pine compared to 33% in the non-native range. For Illumina MiSeq, 5,940 total amplicon sequence variants (ASVs) were detected. P. gigantea represented 14% of the total reads and composed 19% of the reads in the native range and 8% in non-native range of red pine. Furthermore, P. gigantea represented 38% of the reads for stumps that were harvested within 3 years of sampling in the native range of red pine compared to 14% in the non-native range. These results help demonstrate that a higher amount of P. gigantea is present in the native range of red pine and could be acting as a native biological control agent. Additional fungi, including Resinicium bicolor, Hypochnicium cremicolor, Leptographium spp., and others identified at different cutting times are also discussed. Finally, different diversity indices revealed similar, but slightly higher diversity for southern sites via Shannon and Simpson Diversity indices. Beta diversity demonstrated a similar species composition in stumps harvested at different times with these stumps being grouped together based on harvesting years.

Efficacy of Chemical and Biological Stump Treatments for the Control of Heterobasidion occidentale Infection of California Abies concolor

We conducted an experimental evaluation of treatments to limit Heterobasidion occidentale infection of white fir (Abies concolor) stumps and wounds in California mixed conifer forests. We tested the efficacy of urea, borate, and a mixture of two locally collected Phlebiopsis gigantea strains in preventing pathogen colonization of fir stumps and separately, urea and borate as infection controls on experimental stem wounds. These were paired with a laboratory test on ~100 g wood blocks with and without a one-week delay between inoculation and treatment. Urea, borates, and Phlebiopsis treatments all significantly reduced the stump surface area that was colonized by H. occidentale at 84%, 91%, and 68%, respectively, relative to the controls. However, only the borate treatments significantly lowered the number of stumps that were infected by the pathogen. The laboratory study matched the patterns that were found in the stump experiment with a reduced area of colonization for urea, borates, or P. gigantea treatments relative to the controls; delaying the treatment did not affect efficacy. The field wound experiment did not result in any Heterobasidion colonization, even in positive control treatments, rendering the experiment uninformative. Our study suggests treatments that are known to limit Heterobasidion establishment on pine or spruce stumps elsewhere in the world may also be effective on true firs in California.

Comparative Efficacy of State-of-the-Art and New Biological Stump Treatments in Forests Infested by the Native and the Alien Invasive Heterobasidion Species Present in Europe

The Heterobasidion annosum species complex includes major fungal pathogens of conifers worldwide. State-of-the-art preventative stump treatments with urea or with commercial formulations of the fungal biological control agent Phlebiopsis gigantea (i.e., Rotstop®) may become no longer available or are not approved for use in many areas of Europe infested by the three native Heterobasidion species and by the North American invasive H. irregulare, making the development of new treatments timely. The efficacy of Proradix® (based on Pseudomonas protegens strain DSMZ 13134), the cell-free filtrate (CFF) of the same bacterium, a strain of P. gigantea (MUT 6212) collected in the invasion area of H. irregulare in Italy, Rotstop®, and urea was comparatively investigated on a total of 542 stumps of Abies alba, Picea abies, Pinus pinea, and P. sylvestris in forest stands infested by the host-associated Heterobasidion species. Additionally, 139 logs of P. pinea were also treated. Results support the good performances of Rotstop®, and especially of urea against the native Heterobasidion species on stumps of their preferential hosts and, for the first time, towards the invasive North American H. irregulare on stumps of P. pinea. In some experiments, the effectiveness of Proradix® and of the strain of P. gigantea was weak, whereas the CFF of P. protegens strain DSMZ 13134 performed as a valid alternative to urea and Rotstop®. The mechanism of action of this treatment hinges on antibiosis; therefore, further improvements could be possible by identifying the active molecules and/or by optimizing their production. Generally, the performance of the tested treatments is not correlated with the stump size.

Omics analyses and biochemical study of Phlebiopsis gigantea elucidate its degradation strategy of wood extractives

Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based on its extracellular location and our characterization of the recombinant enzyme. Our results provide insight into physiological roles of extractives in the interaction between wood and fungi.

Inter- and Intra-Continental Genetic Variation in the Generalist Conifer Wood Saprobic Fungus Phlebiopsis gigantea

Phlebiopsis gigantea (Fr.) Jülich is a well-known generalist conifer wood saprobe and a biocontrol fungus used in several world countries to prevent stump infection by tree pathogenic Heterobasidion fungal species. Previous studies have reported the presence of regional and continental genetic differentiation in host-specific fungi, but the presence of such differentiation for generalist wood saprobes such as P. gigantea has not been often studied or demonstrated. Additionally, little information exists on the distribution of this fungus in western North America. The main purposes of this study were: (I) to assess the presence of P. gigantea in California, (II) to explore the genetic variability of P. gigantea at the intra and inter-continental levels and (III) to analyze the phylogeographic relationships between American and European populations. Seven loci (nrITS, ML5–ML6, ATP6, RPB1, RPB2, GPD and TEF1-α) from 26 isolates of P. gigantea from coniferous forests in diverse geographic distribution and from different hosts were analyzed in this study together with 45 GenBank sequences. One hundred seventy-four new sequences were generated using either universal or specific primers designed in this study. The mitochondrial ML5–ML6 DNA and ATP6 regions were highly conserved and did not show differences between any of the isolates. Conversely, DNA sequences from the ITS, RPB1, RPB2, GPD and TEF1-α loci were variable among samples. Maximum likelihood analysis of GPD and TEF1-α strongly supported the presences of two different subgroups within the species but without congruence or geographic partition, suggesting the presence of retained ancestral polymorphisms. RPB1 and RPB2 sequences separated European isolates from American ones, while the GPD locus separated western North American samples from eastern North American ones. This study reports the presence of P. gigantea in California for the first time using DNA-based confirmation and identifies two older genetically distinct subspecific groups, as well as three genetically differentiated lineages within the species: one from Europe, one from eastern North America and one from California, with the latter presumably including individuals from the rest of western North America. The genetic differentiation identified here among P. gigantea individuals from coniferous forests from different world regions indicates that European isolates of this fungus should not be used in North America (or vice versa), and, likewise, commercially available eastern North American P. gigantea isolates should not be used in western North America forests. The reported lack of host specificity of P. gigantea was documented by the field survey and further reinforces the need to only use local isolates of this biocontrol fungus, given that genetically distinct exotic genotypes of a broad generalist microbe may easily spread and permanently alter the microbial biodiversity of native forest ecosystems.

Control of Heterobasidion in Norway Spruce Stands: The Impact of Stump Cover on Efficacy of Urea and Phlebiopsis gigantea and Implications for Forest Management

This study investigated the efficacy of Rotstop®, a native Latvian Phlebiopsis gigantea strain and 35% urea solution in combination with a stump cover treatment to control against natural spore infection by Heterobasidion spp. upon precommercial thinning of Norway spruce in three stands growing on former agricultural lands. The major findings were that (i) infection rates of Heterobasidion spp. on stumps treated with the native P. gigantea strain, Rotstop® or urea are similar when stumps are uncovered, and (ii) stump cover promotes stump colonization by the Latvian P. gigantea strain and Rotstop®, leading to a significantly smaller relative area colonized by Heterobasidion spp., as well greater efficiency against Heterobasidion in comparison with urea. Covering of stumps appears beneficial for controlling Heterobasidion stump colonization and may be valuable to forest owners if used in small-scale operations, but it is impractical in automatized thinnings, where managers should consider using regular Rotstop® without covering the stumps.

Phylogenetic Relationships between Phlebiopsis gigantea and Selected Basidiomycota Species Inferred from Partial DNA Sequence of Elongation Factor 1-Alpha Gene

Phlebiopsis gigantea (Fr.) Jülich has been successfully used as a biological control fungus for Heterobasidion annosum (Fr.) Bref., an important pathogen of pine and spruce trees. The P. gigantea species has been known for many years, but our understanding of the relationship between various isolates of this fungus has been substantially improved through the application of DNA sequence comparisons. In this study, relationships between P. gigantea and selected Basidiomycota species was determined, based on elongation factor 1-alpha (EF1α) partial DNA sequence and in silico data. A total of 12 isolates, representing the most representatives of P. gigantea, with diverse geographic distributions and hosts, were included in this study. Phylogenetic trees generated for sequences obtained in this research, grouped the European taxa of P. gigantea and partial sequence of the genome deposed in NCBI database, in a strongly supported clade, basal to the rest of the strains included in the study. P. gigantea isolates originating from Poland, Finland, Sweden, Great Britain and partial sequence of genome formed a monophyletic group. Within this group, isolates of P. gigantea constituted two subclades, showing their partial difference like the two SNPs (single nucleotide polymorphisms) between one and the rest of isolates. The intron and exon relationships among P. gigantea isolates were moreover resolved. The results obtained using the EF1α region should be useful in the selection of more efficient P. gigantea isolates for limiting forest tree root pathogens.