Secondary metabolites and nutrients explain fungal community composition in aspen wood

Fungi are the main decomposers of litter and wood, driving carbon and nutrient cycles. Despite a large number of studies, fungal community composition is remarkably difficult to predict. In the present study, we explore the importance of secondary metabolites and nutrient content in wood and bark as determinants of fungal community composition. We used aspen (Populus tremula) logs of similar size, from one location, and measured concentrations of carbon, nitrogen and secondary metabolites in bark and wood sampled shortly after felling. Fungal DNA was extracted from logs directly after felling and after two seasons of decomposition, and the fungal communities were assessed using DNA-metabarcoding. Concentrations of metabolites varied considerably between individual trees, and we also observed significant differences within single trees. Plant metabolites and nitrogen concentrations significantly affected fungal community composition. For the overall fungal communities and for wood saprotrophic fungi, the explanatory power of wood and bark metabolites was highest in logs decomposed over two seasons. In recently felled trees however, concentration of metabolites had a stronger effect on plant pathogens and endophytes. We conclude that secondary metabolites represent an overlooked, but important niche dimension for fungal communities in both functional sapwood and dead wood.     

Detection of fungal infestations of wood by ion mobility spectrometry

During their growth on wood many fungi produce characteristic volatile organic compounds as secondary metabolites. These microbial volatile organic compounds (MVOCs) can be used as indicators of fungal growth even when such growth is concealed. In order to investigate the formation of these volatile metabolites on building materials, specimens of pine sapwood on agar media colonized by the dry-rot fungus Serpula lacrymans and a mixture of six moulds were examined. MVOCs from this fungal growth were studied over a period of up to half a year by ion mobility spectrometry (IMS) headspace analysis using a sensitive, portable IMS mini-device. The IMS headspace spectra from the growing fungal specimens obtained during this time span are differed from non-incubated wood specimens and indicate the presence of a mixture of MVOCs. The composition and amount of volatile metabolites of the fungi changed during cultivation. This was confirmed by a principal component analysis (PCA). Identification of different MVOCs in the headspace according to drift time and the mobility of ionized gaseous species in reference to GC-MS investigations were proposed. It was concluded that IMS can be used as a rapid and sensitive on-site method to indicate actively growing fungi concealed within wood.     

Structural characterization of metabolites of salvianolic acid B from Salvia miltiorrhiza in normal and antibiotic-treated rats by liquid chromatography-mass spectrometry

This study was conducted to compare the in vivo metabolites of salvianolic acid B (Sal B) between normal rats and antibiotic-treated rats and to clarify the role of intestinal bacteria on the absorption, metabolism and excretion of Sal B. A valid method using LC-MS(n) analysis was established for identification of rat biliary and fecal metabolites. And isolation of normal rat urinary metabolites by repeated column chromatography was applied in this study. Four biliary metabolites and five fecal metabolites in normal rats were identified on the basis of their MS(n) fragmentation patterns. Meanwhile, two normal rat urinary metabolites were firstly identified on the basis of their NMR and MS data. In contrast, no metabolites were detected in antibiotic-treated rat urine and bile, while the prototype of Sal B was found in antibiotic-treated rat feces. The differences of in vivo metabolites between normal rats and antibiotic-treated rats were proposed for the first time. Furthermore, it was indicated that the intestinal bacteria showed an important role on the absorption, metabolism and excretion of Sal B. This investigation provided scientific evidence to infer the active principles responsible for the pharmacological effects of Sal B.     

Metabolism of thromboxane B2 in the monkey.

[3H8]Thromboxane B2 was biosynthesized and infused into an unanesthetized monkey. Several urinary metabolites were isolated and their structures elucidated using gas chromatography-mass spectrometry. In addition to the major urinary metabolite, dinor-thromboxane B2, a series of metabolites resulting from dehydrogenetion of the alcohol group at C-11 were identified: 11-dehydro-thromboxane B2, 11-dehydro-15-keto-13,14-dihydro-2,3-dinor-thromboxane B2, and 11-dehydro-15-keto-13,14-dihydro-19-carboxyl-2,3,4,5-tetranor-thromboxane B2. 6-(1,3-dihydroxypropyl)-7-hydroxy-10-oxo-3-pentadecaenoic acid was also identified. Three mono-O-ethylated metabolites were formed from thromboxane B2, which in this study was infused in an ethanolic solution. A small quantity of thromboxane B2 was excreted unchanged into the urine.     

Aspernolides A and B, butenolides from a marine-derived fungus Aspergillus terreus

Two aromatic butenolides, aspernolides A and B along with the known metabolites, butyrolactone I, terrein and physcion were isolated from the fermentation broth of a soft coral derived fungus Aspergillus terreus. The structures of these metabolites were assigned on the basis of detailed spectroscopic analysis. The absolute stereochemistry of aspernolides A (1) and B (2) was established by their preparation from the known butyrolactone I. Biogenetically aspernolides A and B must be derived from butyrolactone I, a well known specific inhibitor of cyclin dependent kinase (cdk) from A. terreus. When tested, aspernolide A exhibited mild cytotoxicity against cancer cell lines.     

Production of new unsaturated lipids during wood decay by ligninolytic basidiomycetes

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.     

Evaluation of lactic acid bacterium from chilli waste as a potential antifungal agent for wood products

Aims: The aim of this study was to isolate lactic acid bacteria from chilli waste and evaluate metabolites produced for the ability to arrest wood decay. Methods and Results: Using an optical density screening method, one bacterium (isolate C11) was identified as having pronounced antifungal properties against Oligoporus placenta. This isolate was identified as Lactobacillus brevis by 16S rRNA gene sequencing. To determine antifungal activity in wood, Pinus radiata blocks were impregnated with Lact. brevis [C11] cell‐free supernatant and exposed to brown rot fungi O. placenta, Antrodia xantha and Coniophora puteana. The treated timber demonstrated resistance to degradation from all fungi. The antifungal metabolites were heat stable and not affected by proteinase K, but were affected by neutralization with NaOH suggesting the metabolites were of an acidic nature. The presence of lactic and acetic acid was confirmed by HPLC analysis. Conclusions: Lactobacillus brevis [C11] produced acidic metabolites that were able to inhibit the growth of wood decay fungi and subsequent wood decay. Significance and Impact of the Study: Traditional wood treatments are becoming an environmental issue as the public demands more benign options. The use of lactic acid bacteria which are considered safe for general use is a potential alternative to the conventional heavy metal chemicals currently in use.     

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon

Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials.     

Metabolite profiling of Douglas-fir (Pseudotsuga menziesii) field trials reveals strong environmental and weak genetic variation

The primary objective of this study was to assess metabolomics for its capacity to discern biological variation among 10 full-sib families of a Douglas-fir tree breeding population, replicated on two sites. The differential accumulation of small metabolites in developing xylem was examined through metabolite profiles (139 metabolites common to 181 individual trees) generated by gas chromatography mass spectrometry and a series of statistical analyses that incorporated family, site, and tree growth and quantitative phenotypic wood traits (wood density, microfibril angle, wood chemistry and fiber morphology). Multivariate discriminant, canonical discriminant and factor analyses and broad-sense heritabilities revealed that metabolic and phenotypic traits alike were strongly related to site, while similar associations relating to genetic (family) structure were weak in comparison. Canonical correlation analysis subsequently identified correlations between specific phenotypic traits (i.e. tree growth, fibre morphology and wood chemistry) and metabolic traits (i.e. carbohydrate and lignin biosynthetic metabolites), demonstrating a coherent relationship between genetics, metabolism, environmental and phenotypic expression in wood-forming tissue. The association between cambial metabolites and tree phenotype, as revealed by metabolite profiling, demonstrates the value of metabolomics for systems biology approaches to understanding tree growth and secondary cell wall biosynthesis in plants.     

Production of New Unsaturated Lipids during Wood Decay by Ligninolytic Basidiomycetes

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.     

Wood biomechanics and anatomy of PACHYCEREUS PRINGLEI

We report the longitudinal, biomechanical, and anatomical trends observed for tissue samples drawn from the parallel aligned, prismatic woody vascular bundles running the length of a Pachycereus pringlei plant measuring 5.22 m in height. The main vertical stem of this plant was cut into five segments (labeled A through E in the acropetal direction) measuring ～1.02 m in length. Four of the 14 vascular bundles in each segment were surgically removed to obtain 20 vascular bundle segments that were tested in bending to determine their stiffness measured in the radial E(R) and tangential E(T) direction. We also determined the lignin content of representative samples of wood.A nonlinear trend in stiffness was observed: E(R) and E(T) were highest in segments B or C (1.67 GN/m and 1.09 GN/m, respectively), lower in segment A (E(R) = 1.18 GN/m and E(T) = 0.35 GN/m), and lowest in segment E (E(R) = 0.03 GN/m and E(T) = 0.20 GN/m). Similar longitudinal trends were seen for axial tissue volume fraction and fiber wall thickness, which achieved their highest values in segment B (69.8% and 6.59 μm, respectively). Wood stiffness also correlated significantly with cell wall lignin content: with respect to segment B (which had the highest lignin content, and was thus used as the standard reference for percent lignin content), lignin content, was 15, 60, 85, and 43% in segments E, D, C, and A, respectively. Fiber cell length, which increased toward the base of the stem and toward the vascular cambium in the most proximal vascular bundle segment, did not correlate with E(R) or E(T).Basic engineering principles were used to calculate stem stresses resulting from self-loading and any wind-induced bending moment (produced by drag forces). Calculations indicated that the less stiff wood produced in segment A eliminates a rapid and potentially dangerous increase in stresses that would otherwise occur in segments B or C. The less stiff wood in segment A also reduces the probability of shear failure at the cellular interface between the wood and surrounding tissues in this portion of the stem.We conclude that P. pringlei wood stiffness is dependent on the volume fraction and lignification of axial tissues, less so on fiber wall thickness, and that wood development in this species is adaptively responsive to self-loading and differentially applied external mechanical forces.     

New Insights on Wood Dimensional Stability Influenced by Secondary Metabolites: The Case of a Fast-Growing Tropical Species Bagassa guianensis Aubl.

Challenging evaluation of tropical forest biodiversity requires the reporting of taxonomic diversity but also the systematic characterization of wood properties in order to discover new promising species for timber industry. Among wood properties, the dimensional stability is regarded as a major technological characteristic to validate whether a wood species is adapted to commercial uses. Cell structure and organization are known to influence the drying shrinkage making wood density and microfibrils angle markers of choice to predict wood dimensional stability. On the contrary the role of wood extractive content remains unclear. This work focuses on the fast-growing tropical species Bagassa guianensis and we report herein a correlation between heartwood drying shrinkage and extractive content. Chemical extractions and shrinkage experiments were performed on separate wood twin samples to better evaluate correctly how secondary metabolites influence the wood shrinkage behaviour. Extractive content were qualitatively and quantitatively analysed using HPLC and NMR spectroscopy. We found that B guianensis heartwood has a homogeneous low shrinkage along its radius that could not be explained only by its basic density. In fact the low drying shrinkage is correlated to the high extractive content and a corrected model to improve the prediction of wood dimensional stability is presented. Additionally NMR experiments conducted on sapwood and heartwood extracts demonstrate that secondary metabolites biosynthesis occurs in sapwood thus revealing B. guianensis as a Juglans-Type heartwood formation. This work demonstrates that B. guianensis, a fast-growing species associated with high durability and high dimensional stability, is a good candidate for lumber production and commercial purposes.     

IDENTIFICATION OF CELLULOLYTIC BACTERIA ISOLATED FROM THE TERMITE COPTOTERMES CURVIGNATHUS (HOLMGREN)

The aim of the present research was to isolate and identify cellulolytic bacteria from the gut of the local termite Coptotermes curvignathus (Holmgren) present in the vicinity of the University of Putra Malaysia. The isolates were cultured in a medium containing carboxymethyl-cellulose and cellobiose. The bacterial species were tentatively identified by using the Biolog reader as well as the Bergey's manual and later confirmed by 16S rRNA sequence homology. The species were all novel strains and identified as Bacillus cereus strain Razmin A, Enterobacter aerogenes strain Razmin B, Enterobacter cloacae strain Razmin C, Chryseobacterium kwangyangense strain Cb and Acinetobacter strain Raminalimon. Biolog reader was not able to identify one of the bacterial species in which it was identified as C. kwangyangense strain Cb by 16S rRNA sequence homology. The GenBank NCBI/EMBL accession numbers for the bacterial strains are EU294508, EU305608, EU305609, EU169201 and EU332791 for B. cereus Razmin A, E. aerogenes strain Razmin B, E. cloacae strain Razmin C, C. kwangyangense strain Cb and Acinetobacter strain Raminalimon, respectively.

PRACTICAL APPLICATIONS

The experiment describes the isolation and identification of cellulolytic bacteria from termites, using Biolog reader and polymerase chain reaction technique. Termites are common insects in tropical regions and thrive on wood and cellulolytic materials. Hence, the bacteria isolated may be useful in the degradation of cellulosic materials to increase their digestibility and possibly production of metabolites and enzymes.     

Phenolic and heterocyclic metabolite profiles of the grapevine pathogen Eutypa lata

The ascomycete Eutypa lata is the causative agent of eutypa dieback in grapevines, a serious economic problem in major wine grape producing areas. In order to develop a predictive, non-destructive assay for early detection of fungal infection, the phenolic metabolite profiles of 11 strains of E. lata grown on four different artificial growth media were analyzed by HPLC and their variability compared with growth on Cabernet Sauvignon grapevine wood and wood extracts. Six compounds were generally produced in significant amounts, namely eutypinol, eulatachromene, and eutypine and its benzofuran cyclization product, together with siccayne and eulatinol. The two most widely distributed and abundant metabolites were eutypinol and eulatachromene, which were present in 8 of the strains grown on grapewood aqueous extract fortified with sucrose. Metabolite production on grapevine extract was greatly enhanced relative to the artificial media, indicating that this native substrate provides optimal conditions and a more representative profile of the metabolites produced in the natural disease state. The primary metabolites were tested in a grapeleaf disc bioassay to establish their relative toxicity. Neither eutypinol nor siccayne were phytotoxic; eulatachromene, eulatinol, eutypine, and the benzofuran exhibited necrotic effects in the bioassay. The results indicate that eutypa dieback may be caused by several E. lata metabolites rather than a single compound.     

松材线虫对其携带的细菌繁殖的影响

1.引言松材线虫病（Bursaphelenchus xylophilus）是松树的一种毁灭性病害,在日本、中国、韩国和北美、尼日利亚和葡萄牙等国家蔓延,造成了巨大经济损失,其中以日本和中国受害最重.一直认为松材线虫是引起该病的唯一病原,但近十几年来的研究发现,细菌在致病过程中可能起着重要作用,相继从病木和松材线虫体上分离到能对黑松苗有致萎活性的细菌.赵博光等首次根据实验提出松材线虫病是线虫和细菌共同侵染引起的复合侵染病害的假说,并在以后的试验中得到了验证.关于松材线虫对其细菌繁殖的影响研究鲜有报道.本试验采用从感病松树上分离并鉴定了的细菌菌株中选取假单胞属7株、其它属的细菌菌株3株,     

丝状真菌杀松材线虫代谢产物研究进展

松材线虫病是破坏我国森林生态系统最为严重的病害,具有极强的传播性和破坏性,防治此种病害迫在眉睫。基于对物理和化学方式防治松材线虫的研究,对环境友好度最高的生物防治具有更广的研究前景。丝状真菌及其次级代谢产物,来源于自然,与传统的化学杀线虫药剂相比,对环境影响较小,针对松材线虫的致死作用更为专一,因此,从丝状真菌的次级代谢产物中分离获得杀松材线虫活性产物并测定其结构和活性,对于松材线虫病的防治具有重要意义。本文对丝状真菌产生的具有杀松材线虫活性产物的结构、活性展开综述,发现近二十年共有57个活性产物被发现,且结构多种多样,活性差别较大,为了更好地开展此领域的研究,本文对所有产物的结构和活性进行了系统总结,最后又对该领域的研究进行了总结和展望,以期对松材线虫病的生物防治和丝状真菌杀松材线虫次级代谢产物的深入研究提供参考。     

Use of monoclonal antibodies to detect Mn(II)-peroxidase in birch wood degraded by Phanerochaete chrysosporium

Summary A monoclonal antibody (Mab) produced to purified Mn(II)-peroxidase was visualized on and within cell corners of birch wood degraded by Phanerochaete chrysosporium using colloidal gold immuno-transmission electron microscopy techniques. Labelling of the fungal cell membrane and cell wall was also observed. The same Mab was used to visualize the penetration of extracellular fungal metabolite extracts, infiltrated into previously decayed wood. Binding of antibodies to the lignin-rich cell corner region of the middle lamella in wood decayed by P. chrysosporium was observed in sectioned wood blocks and in wood infiltrated with crude extracellular extracts from P. chrysospirium liquid cultures. When a control monoclonal antiserum, produced to extracellular metabolites of Postia (Poria) placenta and cross-reactive with fungal cellulase, was used in labelling, the cellulose rich region of the wood cell walls were labelled. Labelling in the middle lamella cell corners was only noted in what has been described as nonor poorly lignified cell corner regions. Offprint requests to: G. Daniel     

Polyanxanthone A, B and C, three xanthones from the wood trunk of Garcinia polyantha Oliv

Three xanthones, polyanxanthone A (1), B (2) and C (3) have been isolated from the methanol extract of the wood trunk of Garcinia polyantha, along with five known xanthones: 1,3,5-trihydroxyxanthone (4); 1,5-dihydroxyxanthone (5); 1,3,6,7-tetrahydroxyxanthone (6); 1,6-dihydroxy-5-methoxyxanthone (7) and 1,3,5,6-tetrahydroxyxanthone (8). Their structures were determined by means of 1D- and 2D-NMR techniques. Some of the above compounds were screened for their anticholinesterase activity on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes.     

Effects of Saliva on the Viscosity of Gum Solutions

A mesophilic fungal strain Y-94 produced three types of thermostable endo-xylanases accompanied by the formation of a large amount of cellulase. These xylanases were separated from the cellulase by heat treatment at 65°C for 2.5 hr and purified by DEAE-Toyopearl chromatog-raphy, chromatography on an anion exchanger (PBE 94), and Bio-Gel A 0.5 m gel filtration. The molecular weights of the three types of xylanase, designated as xylanase A, B and C, were 51,000, 48,000, and 35,000, respectively. All three enzymes showed highest activity at pH 4.9 and 80°C in lOmin of incubation, and had the same hydrolysis pattern of larch wood xylan with the end- products of xylobiose and xylose. Thus their activities appear essentially the same but not their stabilities. Xylanase A and B were stable from pH 2.5 to 9.0 but xylanase C was unstable above pH 5.5. Xylanase C was unstable at 70°C where other two were stable.     

Inhibition and Stimulation Effects in Communities of Wood Decay Fungi: Exudates from Colonized Wood Influence Growth by Other Species

The effects of exudates from uncolonized and from partly decayed beech wood on the extension rates of 16 later stage decay fungi were investigated. The partly decayed wood had been colonized by the pyrenomycete Eutypa spinosa, or the basidiomycetes Fomes fomentarius, Stereum hirsutum, and Trametes versicolor, all known as common early decay agents in European beech forests. Sterilized wood pieces were placed onto 0.5% malt agar, opposite to small agar plugs containing the test fungi. The latter showed very variable and species-specific growth responses to the various wood types. The presence of uncolonized wood stimulated extension rates in many species, whereas the four previously decayed wood types had variable stimulatory or inhibitory effects. Wood decayed by S. hirsutum resulted in reduced extension rate, delayed growth, or total inhibition in the majority of species, thus it is suggested that this species uses secondary metabolites in a defensive strategy. A single species was, however, stimulated in the presence of S. hirsutum-decayed wood. In contrast, the presence of wood decayed by F. fomentarius was stimulatory to 45% of the species. The other previously decayed wood types generally resulted in more variable responses, depending upon species. The results are discussed in an ecological context and it is suggested that the exudates from the partly decayed wood that are responsible for the reported effects may function as infochemicals, structuring microbial communities in wood.