A comparative study of transgenic canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) harboring either chimeric or native <Emphasis Type="Italic">Chit</Emphasis>42 genes against phytopathogenic fungi

Canola (Brassica napus L.), an agro-economically important crop in the world, is sensitive to many fungal pathogens. One strategy to combat fungal diseases is genetic engineering through transferring genes encoding the pathogenesis-related (PR) proteins such as chitinase which cause the chitin degradation of fungal cell wall. Chitinase Chit42 from Trichoderma atroviride (PTCC5220) plays an important role in biocontrol and has high antifungal activity against a wide range of phytopathogenic fungi. This enzyme lacks a chitin binding domain (ChBD) which is involved in binding activity to insoluble chitin. In the present study, we investigated the effect of chitin binding domain fused to Chit42 when compared with native Chit42. These genes were over-expressed under the CaMV35S promoter in B. napus, R line Hyola 308. Transformation of cotyledonary petioles was achieved by pBISM2 and pBIKE1 constructs containing chimeric and native Chit42 genes respectively, via Agrobacterium method. The insertion of transgenes in T₀ generation was verified through polymerase chain reaction (PCR) and Southern blot analysis. Antifungal activity of expressed chitinase in transgenic plants was also investigated by bioassays. The transgenic canola expressing chimeric chitinase showed stronger inhibition against phytopathogenic fungi that indicates the role of chitin binding domain.     

Identifying the ecological and societal consequences of a decline in <Emphasis Type="Italic">Buxus</Emphasis> forests in Europe and the Caucasus

The potential impact of new invasive tree pests and diseases is usually quantified in economic terms. The ecological and social impacts are less often assessed. Using a comprehensive literature review we assess the potential ecological and social impact of two non-native invasive species (the box tree moth, Cydalima perspectalis and the fungus Calonectria pseudonaviculata) that threaten the survival of box tree, Buxus spp. in forests in Europe and the Caucasus. A total of 132 fungi, 12 chromista (algae), 98 invertebrate and 44 lichens were found to use Buxus spp. Of these, 43 fungi, 3 chromista and 18 invertebrate species have only been recorded on Buxus spp., suggesting that these species are obligate on Buxus spp. and are most at risk from in the loss of Buxus spp. due to these invasive pest and disease species. Buxus spp. was shown to be important for soil stability and water quality but there was no information on other ecosystem functions provided by Buxus spp. Buxus was found to be of considerable historical cultural importance but there was very limited information on current social values and uses. Buxus trees, wood and leaves are associated with different folklore and sacred rites which are still particularly important in the Caucasus. While we could not find any assessment of the economic value of Buxus forests the biodiversity, cultural and social values of Buxus identified here indicate that its loss could have major indirect and non-market economic effects. This work highlights the importance of studying the ecological and societal implications of biological invasions.     

Analysis of the complete genome sequence of the archaeon <Emphasis Type="Italic">Pyrococcus chitonophagus</Emphasis> DSM 10152 (formerly <Emphasis Type="Italic">Thermococcus chitonophagus</Emphasis>)

Here we analyze the first complete genome sequence of Pyrococcus chitonophagus. The archaeon was previously suggested to belong to the Thermococcus rather than the Pyrococcus genus. Whole genome phylogeny as well as whole proteome comparisons using all available complete genomes in Thermococcales clearly showed that the species belongs to the Pyrococcus genus. P. chitonophagus was originally isolated from a hydrothermal vent site and it has been described to effectively degrade chitin debris, and therefore is considered to play a major role in the sea water ecology and metabolic activity of microbial consortia within hot sea water ecosystems. Indeed, an obvious feature of the P. chitonophagus genome is that it carries proteins showing complementary activities for chitin degradation, i.e. endo- and exo-chitinase, diacetylchitobiose deacetylase and exo-β-d glucosaminidase activities. This finding supports the hypothesis that compared to other Thermococcales species P. chitonophagus is adapted to chitin degradation.     

The importance of some microchemical colour reactions in mycology

This work is an examination of the possibilities of the application of microchemical iodine reactions in mycology, of their importance in phylogenesis, and of their practical application for the distinguishing of fungi or moulds from the tissue of the host plant. The best results were obtained with the reaction of chlor-zinc-iodine, then with chlor-calcium-iodine, and with iodophosphoric acid. Reactions were tested with different representatives of developmental groups of fungi and with pure cellulose (cotton-plant fibres) for the comparison of the quantity of cellulose, chitin, or of other substances in the cell-walls. BesidesOomycetes, whose cell-wall consists of cellulose, signs of a “cellulose” reaction appear also inZygomycetes. The species of the orderProtomycetales show a cellulose reaction, which fact also speaks rather for the ranking of this group in the division ofOomycetes. However, the microchemical reaction should not be overestimated, as its colour degree and intensity are influenced by the age of the mycelium of the fungi, by the thickness and physical properties of the cell-wall, and by the presence or absence of the cellular content.     

The role of microorgansisms in maintaining the chitin balance in the Barents Sea

In this study, we investigated chitin hydrolysis by the bacteria inhabiting the ground of the Barents Sea. Four microbial cultures isolated from the ground were described as the genera of Rhodococcus sp., Bacillus sp., Pseudomonas sp., and Acinetobacter sp. Protein complexes with endochitinase and exochitinase activities were purified from the culture liquid. These microorganisms can participate in chitin degradation in sea water. The average molecular weight of the protein fraction with the chitinolytic activity constituted 92–135 kDa. The ratio of the endo-/exochitinase activities of the enzymatic systems was increased in the order Pseudomonas sp. < Bacillus sp. < Acinetobacter sp. < Rhodococcus sp.     

Biodegradation of phenol, <Emphasis Type="Italic">o-</Emphasis>cresol, <Emphasis Type="Italic">m-</Emphasis>cresol and <Emphasis Type="Italic">p-</Emphasis>cresol by indigenous soil fungi in soil contaminated with creosote

Seven non-basidiomycetic fungi, Aspergillus, Candida, Cladosporium, Fusarium, Monicillium, Trichoderma and Penicillium, and two basidiomycetic fungi, Pleurotus and Phanerochaete were isolated from a creosote-contaminated soil by using mineral salts medium and soil extract broth containing antibiotics. Soil contaminated with phenol, o-cresol, m-cresol and p-cresol was collected from the yard of a wood treatment plant in South Africa and inoculated with the strains of Aspergillus, Cladosporium, Fusarium, Monicillium, Penicillium and Phanerochaete, selected from the isolate. The soil in some of the treatment reactors was amended with nutrient supplements to give a C:N:P ratio of 25:5:1. A total of 18 duplicate treatments were established and incubated in the dark at 25°C for 70days. The soil in all the reactors was tilled weekly and moisture was maintained at 70% field capacity. Soil samples were collected every 2weeks for analysis of residual concentrations of the phenols tested, pH measurement and moisture content determination. The nutrient-supplemented treatments were more effective in degrading the phenols (between 84 and 100%) than those that were not supplemented. Barley, which was used as bulking agent enhanced the growth of the fungi and subsequently the degradation of the phenols. Inoculation with a mixture of the six fungal isolates promoted more phenol degradation than with single isolates.     

Overexpression of <Emphasis Type="Italic">PSP1</Emphasis> enhances growth of transgenic Arabidopsis plants under ambient air conditions

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.     

Prospects for the use of new basidiomycete strains for the direct conversion of lignocellulose into ethanol

Sixty-six isolates of basidiomycete fungi were screened for the ability to synthesize cellulase. The effect of temperature on cellulase activity was studied for eight basidiomycete strains as perspective producers of ethanol. The temperature optima of enzyme activity ranged between 26 and 32°C. Direct conversion of Na-carboxymethyl cellulose, microcrystalline cellulose and rye straw were studied for seven basidiomycetes strains: Fomitopsis pinicola MT-5.09, F. pinicola MT-5.21, Piptoporus betulinus MT-30.04, Fomes fomentarius MT-4.05, F. fomentarius MT-4.23, Trametes hirsuta MT-24.24, Flammulina velutipes MT-3.03 Maximum ethanol production from Na-carboxymethyl cellulose (1.3 g/dm³) was achieved by strain F. velutipes MT-3.03. Strain F. fomentarius MT-4.05 more effectively converted rye straw to ethanol with yield of 1.1 g/dm³.     

Genomic insights into the carbohydrate catabolism of <Emphasis Type="Italic">Cairneyella variabilis gen. nov. sp. nov</Emphasis>., the first reports from a genome of an ericoid mycorrhizal fungus from the southern hemisphere

This paper describes a novel species of ericoid mycorrhizal fungus from Australia, Cairneyella variabilis, Midgley and Tran-Dinh, gen. nov. sp. nov. The genome of C. variabilis was sequenced and a draft genome assembled. The draft genome of C. variabilis is 52.4 Mbp in length, and to our knowledge, this is the first study to present a genome of an ericoid mycorrhizal fungus from the southern hemisphere. Using the SignalP and dbCAN bioinformatic pipelines, a study of the catabolic potential of C. variabilis was undertaken and showed genes for an array of degradative enzymes, most of which appear to be secreted from the hyphae, to access a suite of different carbon sources. Isolates of C. variabilis have been previously shown to utilise cellulose, carboxymethyl cellulose (CMC), cellobiose, xylan, pectin, starch and tannic acid for growth, and in the current study, putative enzymes for these processes were revealed. These enzymes likely play key roles in nutrient cycling and other edaphic processes in heathland environments. ITS phylogenetic analyses showed C. variabilis to be distinct from the fungi of the “Hymenoscyphus ericae aggregate”.     

Carbohydrate Specificity of Antibodies against Phytopathogenic Fungi of the <Emphasis Type="Italic">Aspergillus</Emphasis> Genus

Brush rabbits were immunized with injections prepared from the fungi Aspergillus fumigatus, Aspergillus niger, and Aspergillus repens. A library of synthetic biotinylated oligosaccharides containing the key fragments of antigenic polysaccharides of the fungal cell wall—galctomannan, α- and β-glucans, mannan, and chitin—was used to analyze carbohydrate specificity. The anticarbohydrate antibodies obtained from animals immunized with preparations from A. fumigatus and A. repens predominantly recognized epitopes containing galactofuranoside residues, while the majority of the antibodies against A. niger bound the chitooligosaccharide ligand. These results are the basis for the identification of specific markers required for the development of immunoenzyme test systems.     

Black-headed gulls (<Emphasis Type="Italic">Chroicocephalus ridibundus</Emphasis>) - a natural reservoir of potentially pathogenic microfungi?

Mycological studies of selected populations of black-headed gulls were carried out in response to the increasing interest in wild birds as reservoirs of potentially pathogenic fungi and links in the epidemiological chain of mycoses hazardous to human and animals. The biological material comprised swabs from the beaks and cloacae of adult and young birds subjected to standard mycological diagnostics. 79.5% of samples were positive, comprising 22 fungal species belonging to 10 genera, mainly Candida, Rhodotorula, Aspergillus, Fusarium, Cryptococcus, and Trichosporon. The most frequently isolated species were Candida albicans and Rhodotorula rubra, found in the beaks of females and young birds and in the cloacae of young birds with comparable frequency. Cryptococcus laurentii, Cr. neoformans, Aspergillus niger, A. flavus, and Rh. muscilaginosa were isolated relatively frequently from all birds. The results highlight the ecological importance of wild birds in the circulation of potentially pathogenic fungi in the biosphere.     

Alkalitolerant micromycetes in acidic and neutral soils of the temperate zone

A wide diversity of micromycetes from various taxonomic groups in acidic and neutral soils is known from the literature data. In the present work, the fungi isolated from these soils and capable of growth at high pH are analyzed. The fungi were isolated from acidic sod-podzol and neutral cultivated soils by plating on alkaline agar (pH 10.0–10.5). Their identification was carried out using morphological, cultural, and molecular genetic criteria. Phylogenetic analysis was performed and the rates of linear growth within a broad pH range (4.0–10.4) were determined. The isolates represented a polyphyletic group of ascomycetes (Sordariomycetes), which included members of Plectosphaerellaceae (5 species) and various families of Hypocreales (4 species). The most common species were Gibellulopsis nigrescens, Acrostalagmus luteoalbus, Chordomyces antarcticum, and Plectosphaerella spp. Investigation of fungal growth at different pH values revealed all isolates to be alkalitolerant, with no alkaliphilic fungi isolated from acidic sod-podzol and neutral cultivated soils. Although the group of isolates was polyphyletic and its members originated from different ecological and trophic niches, most alkalitolerant isolates exhibited common morphological traits with acremonium- and verticillium-like conidial spore formation, abundant slime formation, and a tendency for aggregation of their mycelium in bundles. Our research confirmed the presence of fungi with alkalitolerant adaptation to external pH in the sod-podzolic and cultivated soils of the Moscow region.     

Biodegradation of lignin by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Q18 and the characterization of a novel bacterial DyP-type peroxidase

Lignin valorization can be obtained through cleavage of selected bonds by microbial enzymes, in which lignin is segregated from cellulose and hemicellulose and abundant phenolic compounds can be provided. In this study, Pseudomonas sp. Q18, previously isolated from rotten wood in China, was used to degrade alkali lignin and raw lignocellulosic material. Gel-permeation chromatography, field-emission scanning electron microscope, and GC–MS were combined to investigate the degradation process. The GC–MS results revealed that the quantities of aromatic compounds with phenol ring from lignin increased significantly after incubation with Pseudomonas sp. Q18, which indicated the degradation of lignin. According to the lignin-derived metabolite analysis, it was proposed that a DyP-type peroxidase (PmDyP) might exist in strain Q18. Thereafter, the gene of PmDyP was cloned and expressed, after which the recombinant PmDyP was purified and the enzymatic kinetics of PmDyP were assayed. According to results, PmDyP showed promising characteristics for lignocellulosic biodegradation in biorefinery.     

Morphological and molecular taxonomy of novel species <Emphasis Type="Italic">Pleurotheciaceae</Emphasis> from freshwater habitats in Yunnan,China

The diversity of lignicolous freshwater fungi of the Greater Mekong Subregion are currently being studied. In this paper, 18 fresh collections of Pleurotheciaceae from submerged wood in freshwater are characterized based on morphological characters and analyses of ITS, LSU, SSU and RPB2 sequence data. Six new species of Pleurotheciella (P. aquatica, P. fusiformis, P. guttulata, P. lunata, P. saprophytica, P. submersa), one new Phaeoisaria species (Ph. aquatica) and one new Pleurothecium species (Pl. aquaticum) are introduced based on their distinct morphology and evidence from molecular phylogeny. Pleurotheciella uniseptata, Phaeoisaria clematidis and Pleurothecium pulneyense are also redescribed and phylogenetic relationships assessed herein, these species are the first records for China.     

Promising cellulolytic fungi isolates for rice straw degradation

The objective of this study was to evaluate the potential of eight fungal isolates obtained from soils in rice crops for straw degradation in situ. From the initial eight isolates, Pleurotus ostreatus T1.1 and Penicillium sp. HC1 were selected for further characterization based on qualitative cellulolytic enzyme production and capacity to use rice straw as a sole carbon source. Subsequently, cellulolytic, xylanolytic, and lignolytic (Pleurotus ostreatus) activity on carboxymethyl cellulose, oat xylan, and rice straw with different nitrogen sources was evaluated. From the results obtained it was concluded both isolates are capable to produce enzymes necessary for rice straw degradation. However, their production is dependent upon carbon and nitrogen source. Last, it was established that Pleurotus ostreatus T1.1 and Penicillium sp. HC1 capability to colonize and mineralize rice straw, in mono-and co-culture, without affecting nitrogen soil content.