The white-rot fungi Phlebia radiata and Dichomitus squalens in wood-based cultures: expression of laccases, lignin peroxidases, and oxalate decarboxylase

Basidiomycetous white-rot fungi are the only organisms that can efficiently decompose all the components of wood. Moreover, white-rot fungi possess the ability to mineralize recalcitrant lignin polymer with their extracellular, oxidative lignin-modifying enzymes (LMEs), i.e. laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP). Within one white-rot fungal species LMEs are typically present as several isozymes encoded by multiple genes. This study focused on two effi cient lignin-degrading white-rot fungal species, Phlebia radiata and Dichomitus squalens. Molecular level knowledge of the LMEs of the Finnish isolate P. radiata FBCC43 (79, ATCC 64658) was complemented with cloning and characterization of a new laccase (Pr-lac2), two new LiP-encoding genes (Pr-lip1, Pr-lip4), and Pr-lip3 gene that has been previously described only at cDNAlevel. Also, two laccase-encoding genes (Ds-lac3, Ds-lac4) of D. squalens were cloned and characterized for the first time. Phylogenetic analysis revealed close evolutionary relationships between the P. radiata LiP isozymes. Distinct protein phylogeny for both P. radiata and D. squalens laccases suggested different physiological functions for the corresponding enzymes. Supplementation of P. radiata liquid culture medium with excess Cu2+ notably increased laccase activity and good fungal growth was achieved in complex medium rich with organic nitrogen. Wood is the natural substrate of lignin-degrading white-rot fungi, supporting production of enzymes and metabolites needed for fungal growth and the breakdown of lignocellulose. In this work, emphasis was on solid-state wood or wood-containing cultures that mimic the natural growth conditions of white-rot fungi. Transcript analyses showed that wood promoted expression of all the presently known LME-encoding genes of P. radiata and laccase-encoding genes of D. squalens. Expression of the studied individual LME-encoding genes of P. radiata and D. squalens was unequal in transcript quantities and apparently time-dependent, thus suggesting the importance of several distinct LMEs within one fungal species. In addition to LMEs, white-rot fungi secrete other compounds that are important in decomposition of wood and lignin. One of these compounds is oxalic acid, which is a common metabolite of wood-rotting fungi. Fungi produce also oxalic-acid degrading enzymes of which the most widespread is oxalate decarboxylase (ODC). However, the role of ODC in fungi is still ambiguous with propositions from regulation of intra and extracellular oxalic acid levels to a function in primary growth and concomitant production of ATP. In this study, intracellular ODC activity was detected in four white-rot fungal species, and D. squalens showed the highest ODC activity upon exposure to oxalic acid. Oxalic acid was the most common organic acid secreted by the ODC-positive white-rot fungi and the only organic acid detected in wood cultures. The ODC-encoding gene Ds-odc was cloned from two strains of D. squalens showing the first characterization of an odc-gene from a white-rot polypore species. Biochemical properties of the D. squalens ODC resembled those described for other basidiomycete ODCs. However, the translated amino acid sequence of Ds-odc has a novel N-terminal primary structure with a repetitive Ala-Ser-rich region of ca 60 amino acid residues in length. Expression of the Ds-odc transcripts suggested a constitutive metabolic role for the corresponding ODC enzyme. According to the results, it is proposed that ODC may have an essential implication for the growth and basic metabolism of wood-decaying fungi.

Degradation of lignin and other 14C-labelled compounds in compost and soil with an emphasis on white-rot fungi

Puu, ruohokasvit ja näistä tehdyt tuotteet kuten mekaanisesta massasta valmistettu sanomalehtipaperi sisältävät ligniiniä, joka hajoaa yleensä hyvin hitaasti luonnossa. Valkolahosienet hajottavat ligniiniä tehokkaimmin, ja koska niiden tuottamat entsyymit hajottavat myös muita vaikeasti hajoavia yhdisteitä, voidaan valkolahosienten avulla mahdollisesti puhdistaa saastuneita maita. Tässä työssä haluttiin selvittää, säilyttävätkö valkolahosienet (Abortiporus biennis, Bjerkandera adusta, Dichomitus squalens, Phanerochaete chrysosporium, Phanerochaete sordida, Phlebia radiata, Pleurotus ostreatus, Trametes hirsuta ja Trametes versicolor) aktiivisuutensa ja kasvavatko ne maassa. Aktiivisuutta mitattiin seuraamalla sienten synteettisen ligniinin (14C-DHP) hajotuskykyä. T. versicolor (silkkivyökääpä) osoittautui tehokkaimmaksi ligniinin hajottajaksi ja sen pentakloorifenolin (PCP) hajotuskykyä tutkittiin erillisessä kokeessa. Entiset tai pitkään käytössä olleet saha-alueet ovat yhä saastuneet puun käsittelyaineista peräisin olevilla kloorifenoleilla. Biohajoavien muovien kehitystyö sekä kompostoinnin yleistyminen jätteiden käsittelymenetelmänä ovat luoneet tarpeen materiaalien biohajoavuuden määrittämiseen. Euroopan standardisoimisjärjestön (CEN) kontrolloidussa kompostitestissä biohajoavuus määritetään materiaalin hajoamisen aikana muodostuvan hiilidioksidin perusteella. Hiilidioksidin tuotto mitataan sekä näytettä sisältävästä kompostista että kompostista ilman näytettä, ja tällöin oletetaan, että kompostin orgaaninen aines molemmissa komposteissa (tausta) tuottaa yhtä paljon hiilidioksidia. Testin puutteeksi saattaa osoittautua kompostissa tai maassa esiintyvä "priming effect". Tällä tarkoitetaan materiaalin lisäämisen jälkeen esiintyvää epänormaalin suurita tai pientä hiilidioksidin muodostusta, minkä seurauksena testin tulosksena saatava biohajoavuus on virheellinen. Ligniinin hajotessa muodostuu enemmän humusta kuin hiilidioksidia, koska ligniini on humuksen tärkein lähtöaine. Näin ollen ligniiniä sisältävät paperituotteet saattavat testin mukaan vaikuttaa biologisesti hajoamattomilta. Valkolahosienet hajottivat 4-23% ligniinistä hiilidioksidiksi ja T. versicolor 29% PCP:sta. Kompostissa ligniini hajosi hiilidioksidiksi 58°C:ssa huomattavasti vähemmän (8%) kuin lämpötiloissa 35°C ja 50°C (23-24%). Kompostin todennäköisesti tärkeimpien ligniinin hajottajien, termofiilisten sienten, tyypillinen optimilämpötila on 45°C, eivätkä ne ole enää aktiivisia 58°C:ssa. Sekä maassa että kompostissa ligniini sitoutui kuitenkin suurimmaksi osaksi humukseen. Valkolahosienet hajottivat sekä humukseen sitoutunutta ligniiniä että PCP:ia, mutta kompostin sekapopulaatio ei tähän pystynyt, ja ligniiniä sitoutui humukseen yhä enemmän kompostoinnin aikana. T. versicolor hajotti PCP:ia tehokkaasti, eikä se tuottanut myrkyllisiä kloorianisoleja, joita jotkut valkolahosienet saattavat muodostaa kloorifenoleista. Priming effect ilmiötä tutkittiin eri ikäisissä ja kypsyydeltään erilaisissa komposteissa. Kompostit erosvat toisistaan myös hajoamattoman jätteen määrän ja mikrobipopulaation suhteen. Negatiivinen priming effect havaittiin kaikissa epästabiileissa komposteissa (ikä enintään 6 kk), ja sen lisäksi yhdessä näistä komposteista positiivinen priming effect kokeen lopussa. Stabiileissa komposteissa (ikä vähintään 6 kk) ilmiötä ei sen sijaan havaittu. Epästabiileissa komposteissa biohajoavuudelle saadut tulokset eivät siis ole luotettavia. Työn tulosten perusteella valkolahosienet, ja erityisesti T. versicolor, ovat lupaavia saastuneen maan puhdistukseen, joskin sienirihmaston mahdollisuudet säilyä aktiivisena maan alkuperäisen mikrobipopulaation kanssa täytyy vielä selvittää. Kompostin sekapopulaatio, joka ei sisällä valkolahosieniä, hajotti ligniiniä yllättävän tehokkaasti termofiilisille sienille sopivissa lämpötiloissa, vaikka ligniini sitoutuikin pääasiallisesti humukseen. Kompostin kypsyys osoittautui tärkeäksi tekijäksi kontrolloidun kompostitestin onnistumisen kannalta. Priming effect ilmiön välttämiseksi on varmistettava, että testissä käytetty komposti on riittävän kypsä. Kompostien mikrobipopulaation koostumusta kompostoinnin eri vaiheissa tulisi tarkemmin selvittää, koska stabiilien ja epästabiilien kompostien ero aiheutui todennäköisesti populaatioiden rakenteessa vallitsevista eroista. Näin myös priming effect ilmiön syyt voitaisiin selittää paremmin.

Growth requirements of some fungi causing maduromycosis

Three strains ofMadurella mycetomi, two ofM. grisea, and two ofRhinocladiella mansonii have been studied for ossible differences in growth requirements which might be used for distinguishing these species. Under the experimental conditions, an incubation temperature of 37C suitedM. mycetomi about as well as 30C.R. mansonii grew less well at 37C than at 30C, andM. grisea did not grow at the higher temperature. M. grisea andR. mansonii further differed fromM. mycetomi in that they required thiamine for growth. The pH tolerance of all the strains was very wide. Asparagine and potassium nitrate were readily utilized by all the strains, but ammonium salts were not. Urea was poorly used byM. mycetomi; the other species did not use it. A possible relationship ofM. grisea andR. mansonii is discusse

Field inoculation with arbuscular-mycorrhizal fungi overcomes phosphorus and zinc deficiencies of linseed (Linum usitatissimum) in a vertisol subject to long-fallow disorder

Long-fallow disorder is expressed as exacerbated deficiencies of phosphorus (P) and/or zinc (Zn) in field crops growing after long periods of weed-free fallow. The hypothesis that arbuscular-mycorrhizal fungi (AMF) improve the P and Zn nutrition, and thereby biomass production and seed yield of linseed (Linum usitatissimum) was tested in a field experiment. A factorial combination of treatments consisting of +/- fumigation, +/- AMF inoculation with Glomus spp., +/- P and +/- Zn fertilisers was used on a long-fallowed vertisol. The use of such methods allowed an absolute comparison of plants growing with and without AMF in the field for the first time in a soil disposed to long-fallow disorder. Plant biomass, height, P and Zn concentrations and contents, boll number and final seed yield were (a) least in fumigated soil with negligible AMF colonisation of the roots, (b) low initially in long-fallow soil but increased with time as AMF colonisation of the roots developed, and (c) greatest in soil inoculated with AMF cultures. The results showed for the first time in the field that inflows of both P and Zn into linseed roots were highly dependent on %AMF-colonisation (R-2 = 0.95 for P and 0.85 for Zn, P < 0.001) in a soil disposed to long-fallow disorder. Relative field mycorrhizal dependencies without and with P+Zn fertiliser were 85 % and 86 % for biomass and 68 % and 52 % for seed yield respectively. This research showed in the field that AMF greatly improved the P and Zn nutrition, biomass production and seed yield of linseed growing in a soil disposed to long-fallow disorder. The level of mycorrhizal colonisation of plants suffering from long-fallow disorder can increase during the growing season resulting in improved plant growth and residual AMF inoculum in the soil, and thus it is important for growers to recognise the cause and not terminate a poor crop prematurely in order to sow another. Other positive management options to reduce long fallows and foster AMF include adoption of conservation tillage and opportunity cropping.

Generalised growth models for aquatic species with an application to blacklip abalone (Haliotis rubra)

This paper presents a maximum likelihood method for estimating growth parameters for an aquatic species that incorporates growth covariates, and takes into consideration multiple tag-recapture data. Individual variability in asymptotic length, age-at-tagging, and measurement error are also considered in the model structure. Using distribution theory, the log-likelihood function is derived under a generalised framework for the von Bertalanffy and Gompertz growth models. Due to the generality of the derivation, covariate effects can be included for both models with seasonality and tagging effects investigated. Method robustness is established via comparison with the Fabens, improved Fabens, James and a non-linear mixed-effects growth models, with the maximum likelihood method performing the best. The method is illustrated further with an application to blacklip abalone (Haliotis rubra) for which a strong growth-retarding tagging effect that persisted for several months was detected. (C) 2013 Elsevier B.V. All rights reserved.

Is stocking barramundi (Lates calcarifer) in north-eastern Queensland a threat to aquatic biodiversity?

The stocking of predators can have significant consequences on recipient aquatic ecosystems. We investigated some potential ecological impacts of stocking a predatory fish (Lates calcarifer) into a coastal river and a large impoundment in north-eastern Australia. L. calcarifer was mostly found in slower-moving, larger reaches of the river or in the main body of the impoundment where there was abundant suitable habitat. In the tidally influenced freshwater reaches of the coastal river, L. calcarifer predominately consumed aytid and palaemonid shrimp that were associated with local macrophyte beds or littoral grasses. In this area the diets of juvenile stocked and wild L. calcarifer were similar and stocked fish displayed a high degree of site fidelity. Further upstream in the river, away from tidal influence, and in the impoundment, fish were the main prey item. Cannibalism was uncommon and we suggest that, at the current stocking densities, there was little dietary evidence of predatory impacts from L. calcarifer on species of conservation concern. We caution against introducing novel predatory species such as L. calcarifer in or near areas that are outside their natural range and are known to support rare, threatened or endangered species.

Online identification guides for Australian smut fungi (Ustilaginomycotina) and rust fungi (Pucciniales)

Interactive identification keys for Australian smut fungi (Ustilaginomycotina and Pucciniomycotina, Microbotryales) and rust fungi (Pucciniomycotina, Pucciniales) are available online at http://collections.daff.qld.gov.au. The keys were built using Lucid software, and facilitate the identification of all known Australian smut fungi (317 species in 37 genera) and 100 rust fungi (from approximately 360 species in 37 genera). The smut and rust keys are illustrated with over 1,600 and 570 images respectively. The keys are designed to assist a wide range of end-users including mycologists, plant health diagnosticians, biosecurity scientists, plant pathologists, and university students. The keys are dynamic and will be regularly updated to include taxonomic changes and incorporate new detections, taxa, distributions and images. Researchers working with Australian smut and rust fungi are encouraged to participate in the on-going development and improvement of these keys.

Precise determination of aquatic plant wet mass using a salad spinner

The reliable assessment of macrophyte biomass is fundamental for ecological research and management of freshwater ecosystems. While dry mass is routinely used to determine aquatic plant biomass, wet (fresh) mass can be more practical. We tested the accuracy and precision of wet mass measurements by using a salad spinner to remove surface water from four macrophyte species differing in growth form and architectural complexity. The salad spinner aided in making precise and accurate wet mass with less than 3% error. There was also little difference between operators, with a user bias estimated to be below 5%. To achieve this level of precision, only 10–20 turns of the salad spinner are needed. Therefore, wet mass of a sample can be determined in less than 1 min. We demonstrated that a salad spinner is a rapid and economical technique to enable precise and accurate macrophyte wet mass measurements and is particularly suitable for experimental work. The method will also be useful for fieldwork in situations when sample sizes are not overly large.

Lipids of some thermophilic fungi

Total lipid content in the thermophilic fungi—Thermoascus aurantiacus, Humicola lanuginosa, Malbranchea pulchella var.sulfurea, andAbsidia ramosa—varied from 5.3 to 19.1% of mycelial dry weight. The neutral and polar lipid fractions accounted for 56.4 to 80.2% and 19.8 to 43.6%, respectively. All the fungi contained monoglycerides, diglycerides, triglycerides, free fatty acids, and sterols in variable amounts. Sterol ester was detected only inA. ramosa. Phosphatide composition was: phosphatidyl choline (15.9–47%), phosphatidyl ethanolamine (23.4–67%), phosphatidyl serine (9.3–17.6%), and phosphatidyl inositol (1.9–11.9%). Diphosphatidyl glycerol occurred in considerable quantity only inH. lanuginosa andM. pulchella var.sulfurea. Phosphatidic acid, detected as a minor component only inM. pulchella var.sulfurea andA. ramosa, does not appear to be a characteristic phosphatide of thermophilic fungi as suggested earlier. The 16∶0, 16∶1, 18∶0, 18∶1, and 18∶2 acids were the main fatty acid components. In addition,A. ramosa contained 18∶3 acid. Total lipids contained an average of 0.93 double bonds per mole of fatty acids, and neutral lipids tend to be more unsaturated than phospholipids.

Communities of wood-inhabiting fungi : Ecological requirements and responses to forest management and fragmentation

Despite much research on forest biodiversity in Fennoscandia, the exact mechanisms of species declines in dead-wood dependent fungi are still poorly understood. In particular, there is only limited information on why certain fungal species have responded negatively to habitat loss and fragmentation, while others have not. Understanding the mechanisms behind species declines would be essential for the design and development of ecologically effective and scientifically informed conservation measures, and management practices that would promote biodiversity in production forests. In this thesis I study the ecology of polypores and their responses to forest management, with a particular focus on why some species have declined more than others. The data considered in the thesis comprise altogether 98,318 dead-wood objects, with 43,085 observations of 174 fungal species. Out of these, 1,964 observations represent 58 red-listed species. The data were collected from 496 sites, including woodland key habitats, clear-cuts with retention trees, mature managed forests, and natural or natural-like forests in southern Finland and Russian Karelia. I show that the most relevant way of measuring resource availability can differ to a great extent between species seemingly sharing the same resources. It is thus critical to measure the availability of resources in a way that takes into account the ecological requirements of the species. The results show that connectivity at the local, landscape and regional scales is important especially for the highly specialized species, many of which are also red-listed. Habitat loss and fragmentation affect not only species diversity but also the relative abundances of the species and, consequently, species interactions and fungal successional pathways. Changes in species distributions and abundances are likely to affect the food chains in which wood-inhabiting fungi are involved, and thus the functioning of the whole forest ecosystem. The findings of my thesis highlight the importance of protecting well-connected, large and high-quality forest areas to maintain forest biodiversity. Small habitat patches distributed across the landscape are likely to contribute only marginally to protection of red-listed species, especially if habitat quality is not substantially higher than in ordinary managed forest, as is the case with woodland key habitats. Key habitats might supplement the forest protection network if they were delineated larger and if harvesting of individual trees was prohibited in them. Taking the landscape perspective into account in the design and development of conservation measures is critical while striving to halt the decline of forest biodiversity in an ecologically effective manner.