Wolfiporia Cocos Research Articles

Occurrence, distribution and estimated intake of mercury and selenium from sclerotia of the medicinal fungus Wolfiporia cocos from China

The contamination and distribution of mercury and selenium in the Chinese medicinal fungus Wolfiporia cocos was investigated. The sclerotial mercury concentrations ranged from 0.0043 to 0.027 mg kg1 dry biomass (db) in the inner white part and 0.019–0.074 mg kg−1 db in the shell (outer part), while selenium concentrations ranged from < 0.00048 to 0.0040 mg kg−1 db (white) and 0.0034–0.038 mg kg−1 db (shell). Positive correlations were found for mercury, as well as for mercury and selenium but they were not consistent for both morphological parts. Mercury concentrations exceeded selenium in 16 of 17 white part pools (molar quotient 0.53 to > 10) and in 11 of 17 shell pools (quotient 0.37 to 3.2). The estimated maximal exposure to mercury contained in sclerotial products based on 45 g per capita daily intake for a 60 kg individual over one week, was 0.000020 mg kg−1 body mass (bm; white) and 0.000055 mg kg−1 bm (shell) on a daily basis, and 0.00014 mg kg−1 bm (white) and 0.00039 mg kg−1 bm (shell) on a weekly basis. Relative to mercury, the corresponding intake rates of selenium were considered very low, i.e., they averaged on a daily basis at 0.00075 μg kg−1 bm (white) and 0.0097 μg kg−1 bm (shell) with maximum intake at 0.0030 μg kg−1 bm (white) and 0.028 μg kg−1 bm (shell).

Lanostane Triterpenoids with Glucose-Uptake-Stimulatory Activity from Peels of the Cultivated Edible Mushroom Wolfiporia cocos.

A chemical study on the peels of the cultivated edible mushroom Wolfiporia cocos led to the isolation and identification of 47 lanostane triterpenoids including 16 new compounds (1-16). The structures of the new compounds were determined by analysis of the NMR, MS, and electronic circular dichroism (ECD) data. Compounds 1 and 2 represent new members of the family of 4,5-secolanostane triterpenes. Compound 3 is a new aromatic lanostane triterpene with an unusual methyl rearrangement from C-10 to C-6. The absolute configurations of 1 and 8 were assigned by ECD spectra calculation. All compounds were evaluated for cytotoxicity (K562, SW480, and HepG2) and glucose-uptake-stimulating effects. Compounds 23, 25, 29, and 31 showed weak inhibition on the K562 cells with IC50 in the range of 25.7 to 68.2 μM, respectively. Compounds 21, 28, and 30 increased the glucose uptake in 3T3-L1 cells by 25%, 14%, and 50% at 5 μM, respectively. In addition, compounds 14, 23, 29, 35, and 43 showed insulin-sensitizing activity by increasing the insulin-stimulated glucose uptake at 2.5 μM in 3T3-L1 adipocytes. A preliminary structure-activity relationship analysis indicates that the 6/6/6/5 ring skeleton and the double bond between C-8 and C-9 are beneficial for the glucose-uptake-stimulating and insulin-sensitizing activities. Furthermore, the alkaline-insoluble fraction mainly containing compounds 22, 24, 28, and 31 were confirmed to have hypoglycemic and hypolipidemic activity on high-fat-diet-induced obese mice. This work confirms the potential of the peels' extracts of W. cocos as a functional food or dietary supplements.

The complete mitochondrial genome of Wolfiporia cocos (Polypolales: Polyporaceae)

Wolfiporia cocos have been used as diuretic, sedative, and tonic medicine. Although its medicinal effects were reported, its genomic information is rare. In this paper, we report the complete mitochondrial genome of Wolfiporia cocos which is a 136,067 bp circular structure with 5 protein-coding genes, 25 tRNA genes, and 2 rRNA genes. Additional six protein-coding genes (cob, cox1, cox2, cox3, nad2, nad6) were truncated or pseudogenized by internal stop codons. Phylogenetic analysis of the mitochondrial genome of W. cocos showed the distinct relationship with other five mitochondrial genomes of Polyporales.

Determination of the Five Main Terpenoids in Different Tissues of Wolfiporia cocos.

Wolfiporia cocos is a fungus containing triterpenoids and is widely used as an herbal medicine. However, it is unknown whether its main triterpenoid contents differ in different tissues. In this study, we identified dehydrotumulosic acid, polyporenic acid C, pachymic acid, dehydrotrametenolic acid, and dehydroeburicoic acid as the five main triterpenoids in W. cocos. We also systematically profiled the contents and distribution of these main triterpenoids in different tissues of W. cocos. High contents of all five triterpenoids were found in the surface layer of W. cocos. Intriguingly, we noted that the highest contents of the five triterpenoids were found in the surface layer of the sclerotium grown under pollution-controlled cultivation; the second-highest contents were found in the surface layer of the natural sclerotium. These results indicate that environmentally friendly cultivation of the sclerotium of W. cocos is a practical way to increase the productivity of W. cocos. In addition, our findings suggest that the triterpenoids may contribute to the pharmacological activity of W. cocos, and the surface layer of sclerotium in W. cocos might be a promising raw material for applications in health care and the development of functional medical products.

Evaluation on intrinsic quality of Wolfiporia cocos influenced by cultivation methods by a multi-step IR macro-fingerprinting technique

Wolfiporia cocos is a well-known traditional East-Asian medicinal fungus. In this study, four Wolfiporia cocos samples (FC-DM, RC-DM, FC-YN and SC-YN) of three cultivation methods harvest from Dabie Mountain area and Yunnan province were discriminated and identified by a multi-step IR macro-fingerprinting technique. The correlation coefficient of the three cultivation methods of Wolfiporia cocos are higher than 0.98 in the range of 450–2600 cm−1. According to retrieving the spectrum in Sadtler database, the main compositions of Wolfiporia cocos are laminarin polysaccharide. While Correlation coefficients of synchronous 2D-IR spectra of FC-DM and other Wolfiporia cocos samples were furtherly calculated, acquiring a quantitative discrimination result. The thermal sensitivity of polysaccharides of SC-YN is different from other samples in the range of 850–1370 cm−1. The difference in thermal sensitivity of CH (1402 and 1474 cm−1) and amino acid (1666 or 1651 cm−1) could also be considered as the key factors to discriminate them. The result proved that the 2DCOS-IR could discriminate Wolfiporia cocos of different cultivation methods.

Intracellular Lipid Accumulation Inhibition, Anticancer Activity, and Single Oral Dose Toxicity of Ethanolic Wolfiporia cocos Extracts

Intracellular Lipid Accumulation Inhibition, Anticancer Activity, and Single Oral Dose Toxicity of Ethanolic Wolfiporia cocos Extracts

De Novo Sequencing of a Sparassis latifolia Genome and Its Associated Comparative Analyses.

Known to be rich in β-glucan, Sparassis latifolia (S. latifolia) is a valuable edible fungus cultivated in East Asia. A few studies have suggested that S. latifolia is effective on antidiabetic, antihypertension, antitumor, and antiallergen medications. However, it is still unclear genetically why the fungus has these medical effects, which has become a key bottleneck for its further applications. To provide a better understanding of this fungus, we sequenced its whole genome, which has a total size of 48.13 megabases (Mb) and contains 12,471 predicted gene models. We then performed comparative and phylogenetic analyses, which indicate that S. latifolia is closely related to a few species in the antrodia clade including Fomitopsis pinicola, Wolfiporia cocos, Postia placenta, and Antrodia sinuosa. Finally, we annotated the predicted genes. Interestingly, the S. latifolia genome encodes most enzymes involved in carbohydrate and glycoconjugate metabolism and is also enriched in genes encoding enzymes critical to secondary metabolite biosynthesis and involved in indole, terpene, and type I polyketide pathways. As a conclusion, the genome content of S. latifolia sheds light on its genetic basis of the reported medicinal properties and could also be used as a reference genome for comparative studies on fungi.

Elemental characteristics of mushroom species cultivated in China and Poland

China is the first and Poland the fifth the greatest producers of cultivated mushrooms worldwide. Because new species are being domesticated and gaining popularity there is a need to study their biological activities and chemical composition. In this study, a multi-elemental analysis was conducted on 14 culinary and/or medicinal mushroom species cultivated in China and Poland, all of which were available in German and Polish oriental and internet shops (Agaricus bisporus, Amauroderma rude, Auricularia auricula-judae, Auricularia nigricans, Ganoderma lucidum, Lentinula edodes, Lignosus rhinocerus, Ophiocordyceps sinensis, Pleurotus ostreatus, Sparassis crispa, Tremella fuciformis, Wolfiporia cocos and Volvariella volvacea). Overall, the contents of 5 macroelements and 31 trace elements were quantified. The studied mushrooms varied widely in their content of both essential and toxic deleterious elements. A. rude was found to contain the highest content of toxic deleterious elements Al, As and Pt. Platinum content was of particular concern as it exceeded 7 mg kgö1 dry matter. The studied mushrooms were generally found to contain higher amounts of Pt, Ni (particularly W. cocos), and rare-earth elements Er and Nd (particularly V. volvacea) than reported in many literature data. The study generally concludes that the levels of various elements in the analysed mushrooms are not toxic, although further attention should be paid to reducing Ni levels in commercial mushrooms available as culinary.

The Phosphatome of Medicinal and Edible Fungus Wolfiporia cocos.

Wolfiporia cocos is an important medicinal and edible fungus that grows in association with pine trees, and its dried sclerotium has been used as a traditional medicine in China for centuries. However, the commercial production of W. cocos sclerotia is currently limited by shortages in pine wood resources. Since protein phosphatases (PPs) play significant roles in growth, signal transduction, development, metabolism, sexual reproduction, cell cycle, and environmental stress responses in fungi, the phosphatome of W. cocos was analyzed in this study by identifying PP genes, studying transcript profiles and assigning PPs to orthologous groups. Fifty-four putative PP genes were putatively identified in W. cocos genome based on homologous sequences searching using BLASTx program against the Saccharomyces cerevisiae, Fusarium graminearum, and Sclerotinia sclerotiorum databases. Based on known and presumed functions of orthologues of these PP genes found in other fungi, the putative roles of these W. cocos PPs in colonization, hyphal growth, sclerotial formation, secondary metabolism, and stress tolerance to environment were discussed in this study. And the level of transcripts from PP genes in the mycelium and sclerotium stages was also analyzed by qRT-PCR. Our study firstly identified and functional discussed the phosphatome in the medicinal and edible fungus W. cocos. The data from our study contribute to a better understanding of PPs potential roles in various cellar processes of W. cocos, and systematically provide comprehensive and novel insights into W. cocos economically important traits that could be extended to other fungi.

Morphological characteristics of fruit bodies and basidiospores of Wolfiporia extensa

Wolfiporia cocos is a well-known traditional medicine in China, Japan, Korea, and other Asian countries owing to its numerous therapeutic properties. With the aim to determine the morphology and genetic characteristics of W. cocosten strains of W. cocos were cultivated in vitro, and subsequently, rapid amplification of polymorphic DNA was performed. To the best of our knowledge, this is the first study to examine the morphology of fruit bodies of W. cocos in Korea. W. cocos were cultured on PDA agar at different temperatures (12, 16, 20, 24, and 28oC) under 12-hour light (600 Lux) / 12-hour dark photoperiod condition for 1 month. Appearance of fruit body was the highest at 28°C condition in all the strains investigated. Honeycomb-like structure on sclerotia was observed in Andong 01, Andong 02, Andong 03, KFRI 1104, KFRI 1105, KFRI 1106, KFRI 1107, KFRI 1108, and ASI 13007 strains of . The KFRI 1103 strain formed cosmos petal-like structure on sclerotia. The average size of basidiospores was recorded as 7.55 μm in height and 3.35 μ in width.

The Kinome of Edible and Medicinal Fungus Wolfiporia cocos

Wolfiporia cocos is an edible and medicinal fungus that grows in association with pine trees, and its dried sclerotium, known as Fuling in China, has been used as a traditional medicine in East Asian countries for centuries. Nearly 10% of the traditional Chinese medicinal preparations contain W. cocos. Currently, the commercial production of Fuling is limited because of the lack of pine-based substrate and paucity of knowledge about the sclerotial development of the fungus. Since protein kinase (PKs) play significant roles in the regulation of growth, development, reproduction, and environmental responses in filamentous fungi, the kinome of W. cocos was analyzed by identifying the PKs genes, studying transcript profiles and assigning PKs to orthologous groups. Of the 10 putative PKs, 11 encode atypical PKs, and 13, 10, 2, 22, and 11 could encoded PKs from the AGC, CAMK, CK, CMGC, STE, and TLK Groups, respectively. The level of transcripts from PK genes associated with sclerotia formation in the mycelium and sclerotium stages were analyzed by qRT-PCR. Based on the functions of the orthologs in Sclerotinia sclerotiorum (a sclerotia-formation fungus) and Saccharomyces cerevisiae, the potential roles of these W. cocos PKs were assigned. To the best of our knowledge, our study is the first identification and functional discussion of the kinome in the edible and medicinal fungus W. cocos. Our study systematically suggests potential roles of W. cocos PKs and provide comprehensive and novel insights into W. cocos sclerotial development and other economically important traits. Additionally, based on our result, genetic engineering can be employed for over expression or interference of some significant PKs genes to promote sclerotial growth and the accumulation of active compounds.

De novo assembly and transcriptome analysis of sclerotial development in Wolfiporia cocos

Wolfiporia cocos Ryvarden et Gilbertson, a well-known medicinal fungus in the Basidiomycetes, is widely distributed in East Asia. Its dried sclerotium, which is known as Fuling in China, has been used as a traditional crude drug in Chinese traditional medicine for thousand years. However, little is known about how the sclerotium is developed at the genetic level. In this study, the de novo sequencing of sclerotia of W. cocos (S1_initial stage; S2_developmental stage and S3_mature stage) was carried out by illumina HiSeq 2000 technology. 27,438 unigenes were assembled from ~30Gbp raw data, and 12,093 unigenes were significantly annotated. The analysis of expression profiles during development returned 304 differentially expressed genes (DEGs), which were clustered into four different groups according to their expression trends. Especially for the maturation stage (S3), the sclerotium exhibited a markedly different expression profile from other stages. We further showed that peroxisome, unsaturation of fatty acids and degradation pathway were respectively prevalent in S1, S2 and S3 stages as evidenced by enrichment analysis. To our knowledge, this study represents the first report of sclerotial development transcriptomics in W. cocos. The obtained results provide novel insights into the developmental biology of the sclerotia, which is helpful for future studies about cultivation and breeding of W. cocos.

Development of crossbreeding high-yield-potential strains for commercial cultivation in the medicinal mushroom Wolfiporia cocos (Higher Basidiomycetes).

Wolfiporia cocos is a well-known medicinal mushroom, and its dried sclerotia has been widely used as a traditional medicine in China, Japan, and other Asian countries for centuries. However, long-term asexual reproduction of the breeding system in W. cocos results in a current universal degeneration of cultivated strains. To develop a W. cocos breeding program that will benefit commercial cultivation, we previously developed an optimum method for indoor induction of W. cocos fruiting bodies and clarified the nature of preponderant binuclear sexual basidiospores. In this paper, we first show that the majority of W. cocos single-spore isolates cannot form sclerotium in field cultivation. We then investigated the possibility of breeding new strains by crossbreeding. Three types of mating reactions were observed in both intra-strain pairings and inter-strain pairings, and a total of fifty-five hybrids were selected by antagonistic testing and allele-specific polymerase chain reaction (PCR). Field cultivation of hybrids demonstrated that some hybrids can form sclerotium via two cultivated methods. Two new high-yield strains were identified. This report will stimulate new thinking on W. cocos and promote further extensive studies on crossbreeding in W. cocos, a new topic related to the development of more efficient protocols for the discrimination of hybrids in W. cocos.

Genetic diversity of Chinese Wolfiporia cocos cultivars revealed by phenotypic traits and ISSR markers

Wolfiporia cocos, a well-known medicinal fungus, has been widely used in China and other Asian countries due to its various therapeutic value. In this study, genetic diversity among 22 Chinese W. cocos cultivars were assessed using phenotypic traits and inter-simple sequence repeat (ISSR). The results demonstrated that a total of 686 bands were amplified by 17 selected primers, 81.9 % of which were found to be polymorphic. Genetic distance was from 1.05 to 7.62 (physiological characters), and the similarity coefficients were from 0.63 to 0.82 (ISSR). Both methods were effective in differentiating among strains. The unweighted pair-group method with arithmetic averages (UPGMA) dendrogram classified the 22 strains into six major groups. Comprehensive analysis indicated that the cultivated germplasm of W. cocos in China were rich in genetic diversity. The results of this study has contributed substantially to our knowledge of W. cocos genetic diversity and also provides a theoretical basis of use and conservation for W. cocos resource and breeding.

Comparing lignocellulose physiochemistry after decomposition by brown rot fungi with distinct evolutionary origins.

Among wood-degrading fungi, lineages holding taxa that selectively metabolize carbohydrates without significant lignin removal (brown rot) are polyphyletic, having evolved multiple times from lignin-removing white rot fungi. Given the qualitative nature of the 'brown rot' classifier, we aimed to quantify and compare the temporal sequence of carbohydrate removal among brown rot clades. Lignocellulose deconstruction was compared among fungi using distinct plant substrates (angiosperm, conifer, grass). Specifically, aspen, pine and corn stalk were harvested over a 16-week time series from microcosms containing Gloeophyllum trabeum, Fomitopsis pinicola, Ossicaulis lignatilis, Fistulina hepatica, Serpula lacrymans, Wolfiporia cocos or Dacryopinax sp. After quantifying plant mass loss, a thorough compositional analysis was complemented by a saccharification test to determine wood cell wall accessibility. Mass loss and accessibility varied depending on fungal decomposer and substrate, and trajectories of loss for hemicellulosic components and cellulose differed among plant tissue types. At any given stage of decomposition, however, lignocellulose accessibility and the fraction remaining of carbohydrates and lignin within a plant tissue type were generally the same, regardless of fungal isolate. This suggests that the sequence of plant component removal at this typical scale of characterization is shared among these brown rot lineages, despite their diverse genomes and secretomes.

Effect of revulsive cultivation on the yield and quality of newly formed sclerotia in medicinal Wolfiporia cocos.

Wolfiporia cocos is a well-known medicinal mushroom widely used in China, Japan and other Asiatic countries for its various therapeutic effects. 'Revulsive cultivation' is a newly developed method for promoting sclerotia growth in W. cocos field cultivation in China. In this report, we have systematically examined the effects of 'revulsive cultivation' on the yield and quality of newly formed sclerotia. The results showed that the genetic differences between the cultivated strain and the revulsive strain of T1 used in this study did not affect the formation process of new, large sclerotia in which the mycelia of the cultivated strain grew on pine logs directionally assembled on the revulsive strain. Additionally, 'revulsive cultivation', in which the cultivated strain and the revulsive strain used had the same or different genotypes, could remarkably increase the yield, lower the water content, and increase the water-soluble polysaccharide content of the newly formed sclerotia. Moreover, we observed that the changes in the values of the tested economic traits obtained from different genotype combinations through 'revulsive cultivation' were dissimilar. The correlations of these changes with the original sclerotium-forming ability of the cultivated strains and the genetic differences between the cultivated strain and the revulsive strain were not significant. These results will broaden our knowledge regarding the field cultivation of this medical fungus, stimulate new thinking on the study of sclerotium formation in some sclerotium-forming fungi, and promote further studies on the mechanism of sclerotium formation in W. cocos.

CELL DETACHMENT BY PROLYL-SPECIFIC ENDOPEPTIDASE FROM &lt;i&gt;WOLFIPORIA COCOS&lt;/i&gt;

As requirements for Advanced Therapy Medicinal Product (ATMP) production differ from other production processes (e.g., therapeutic protein production), cell detachment is often a crucial step for the process success. In most cases, cell detachment is done enzymatically. Although many peptidases are established in cell culture in R&D, e.g., Trypsin as gold standard, many of them seem to be unsuitable in ATMP production processes. Therefore, the present study investigated a novel endopeptidase used in food biotechnology for its applicability in ATMP processes where cell detachment is needed. The Prolyl-specific Peptidase (PsP) is of non-mammalian origin and considered as safe for humans. PsP was purified from the supernatant of the fungus Wolfiporia cocos. The isolation and purification resulted in an enzyme solution with 0.19 U mg-1 prolyl-specific activity. By in silico analysis it was confirmed that attachment-promoting proteins can be cleaved by PsP in a similar amount than with Trypsin. Further the proteolytic activity was determined for PsP and Trypsin by using the same enzymatic assay. Detachment with both enzymes was compared for cells used in typical therapeutic production processes namely a mesenchymal stem cell line (hMSC-TERT) as a model for a cell therapeutic, Vero and MA104 cells used for viral therapeutic or vaccine production. The cell detachment experiments were performed with comparable enzyme activities (1.6 U mL-1). hMSC-TERT detachment was faster with PsP than with Trypsin. For Vero cells the detachment with PsP was not only faster but also more efficient. For MA104 cells the detachment rate with PsP was similar to Trypsin. For all cell types, detachment with PsP showed less influence on cell growth and metabolism compared to standard Trypsin.Thus, three cell types used in ATMP, viral therapeutics or vaccine production can be detached efficiently and gently with PsP. Therefore, PsP shows potential for cell detachment in ATMP and viral/vaccine production processes.

Polyporales genomes reveal the genetic architecture underlying tetrapolar and bipolar mating systems

The process of mating in Basidiomycota is regulated by homeodomain-encoding genes (HD) and pheromones and G protein-coupled pheromone receptor genes (P/R). Whether these genes are actually involved in determining mating type distinguishes mating systems that are considered tetrapolar (two locus) from bipolar (one locus). Polyporales are a diverse group of wood-decay basidiomycetes displaying high variability in mating and decay systems. Many of the bipolar species appear to be brown-rot fungi, and it has been hypothesized that there is a functional basis for this correlation. Here we characterize mating genes in recently sequenced Polyporales and other Agaricomycete genomes. All Agaricomycete genomes encode HD and pheromone receptor genes regardless of whether they are bipolar or tetrapolar. The HD genes are organized into a MAT-HD locus with a high degree of gene order conservation among neighboring genes, with the gene encoding mitochondrial intermediate peptidase consistently syntenic but no linkage to the P/R genes. To have a complete dataset of species with known mating systems we determined that Wolfiporia cocos appears to be bipolar, using the criterion that DNA polymorphism of MAT genes should be extreme. Testing the correlation of mating and decay systems while controlling for phylogenetic relatedness failed to identify a statistical association, likely due to the small number of taxa employed. Using a phylogenetic analysis of Ste3 proteins, we identified clades of sequences that contain no known mating type-specific receptors and therefore might have evolved novel functions. The data are consistent with multiple origins of bipolarity within the Agaricomycetes and Polyporales, although the alternative hypothesis that tetrapolarity and bipolarity are reversible states needs better testing.

Ancient Chinese People’s Knowledge of Macrofungi during the Period from 220 to 589

To explore the knowledge of macrofungi in ancient China from 220 to 589, several aspects are studied in detail, including the meanings of ancient Chinese characters or words for macrofungi, naturalistic descrip­tions of macrofungi, the earliest record of the cultivation of macrofungi, the earliest record of the custom of eating macrofungi in Taiwan Island, methods of cooking macrofungi dishes, macrofungi in literature, and macrofungi in Taoist texts. More kinds of macrofungi are mentioned in the various works of this period than in earlier works, including the earliest record of the cultivation of fuling 茯苓 ( Wolfiporia cocos ), as well as the earliest record of the custom of eating monkey head mushroom ( houtou gu 猴頭菇; Hericium erinaceus ). There are two intriguing Taoist monographs of great significance for the history of zhi 芝 in Taoist literature. Moreover, the stories about lingzhi 靈芝 ( Ganoderma lucidum ) in Chinese literature indicate that macrofungi were not only lauded by poets, but also used by them to represent virtues.

Choice tests and neighbor effects during fungal brown rot of copper- and non-treated wood

In previous work, a ‘choice’ test was used to test wood durability in mixtures of wood types. Results demonstrated that a filamentous fungus (Gloeophyllum trabeum) made a ‘choice,’ degrading white pine significantly less when less-durable woods were readily available. For this experiment, we substituted white pine with copper-treated pine and used three fungi (G. trabeum, Serpula himantioides, and Wolfiporia cocos) representing a range of copper tolerances. Southern pine blocks treated commercially with micronized copper azole or treated in-house using copper ethanolamine were used as treated wood substrates. Trials were soil-block tests, using two-block combinations to include non-treated, non-durable pine or as single-block treatments. After 12 weeks of decay, copper tolerance by S. himantioides and W. cocos was evident while G. trabeum was not copper tolerant. Availability of non-durable pine blocks, however, had no effect on decay in copper-treated wood. This contrasts with our past results using white pine. The reason for the discrepancy may relate to fungal mechanisms of copper tolerance (e.g., constitutive oxalic acid secretion) which may differ from mechanisms to overcome extractives in white pine. More fungal research using choice trials to assess these ‘neighbor effects’ is logical, given increased use of mixed, composite, and hybrid materials.