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PubMed2005

Characterisation of selenium compounds in rye seedling biomass using 75Se-labelling/SDS-PAGE separation/gamma-scintillation counting, and HPLC-ICP-MS analysis of a range of enzymatic digests.

In the present study, selenium-enriched plant biomass was investigated to evaluate the ability of rye seedlings to take up, and assimilate, inorganic selenium. Two different analytical approaches were used. Electrophoretic separation (SDS-PAGE) of proteins extracted from 75Se-labelled biomass was used to investigate the biotransformation of selenite into organic forms of the element. Ion-pair chromatography coupled with ICP-MS detection was chosen for the analysis of selenium species, enzymatically extracted from the plant biomass. The results of three enzymatic hydrolysis procedures and three sequential enzymatic extractions procedures are compared. The most effective single extraction was proteolysis (using protease type XIV), giving an overall extraction efficiency of 48%. However, for combinations of enzymes, the most effective was cellulase (Trichoderma viride) followed by sequential extraction of the solid pellet using protease type XIV, giving an extraction efficiency of 70%. The complementary data from the electrophoretic fractionation of proteins, and the HPLC separation of Se-species in the proteolytic digests, reveal the existence of large number of selenium-containing compounds in the rye seedling plant biomass. The results showed the complete biotransformation of inorganic selenium into organic forms during germination of the rye seedlings. HPLC-ICP-MS analysis of extracts from the plant biomass did not show the presence of selenate or selenite. At the time of this study, the lack of suitable organic-MS facilities meant that it was not possible to characterise them fully. However, the data does show that a combination of different enzymes, rather than just the commonly-used protease, should be considered when developing an extraction strategy for selenium in different food types to those already reported in the literature.

Biomass↗

PubMed2002

Effectiveness of municipal waste compost and its humic fraction in suppressing Pythium ultimum.

The effect of addition of a municipal solid waste (MSW) compost and its water-soluble and humic fraction to suppress the effect of Pythium ultimum on pea plants was studied and compared with that of a chemical pesticide (metalaxyl). The biotic and abiotic characteristics of compost involved in the biocontrol effects of these materials were also evaluated. The addition into soil of whole composts and their humic fractions reduced the effect of the pathogen on pea plants, significantly reducing the number of root lesions and Pythium populations and avoiding reductions of plant growth. The greatest pathogen suppression was achieved with the chemical pesticide. However, it also caused a significant decrease in the number of nontarget bacteria and fungi and on beneficial soil microorganisms such as Trichoderma and Pseudomonas. Addition of organic amendments increased population size of nontarget and specific biocontrol microorganisms. The humic fraction showed similar results to compost. All this suggests that metalaxyl has a nonspecific effect, producing adverse effects on aspects of soil quality. This was avoided if the chemical pesticide was reduced and replaced by organic amendments such as an MSW compost or its humic fraction.

Agriculture↗

PubMed2001

Soil Saprotrophic Microfungi Associated with Roots of Calamagrostis epigeios on an Abandoned Deposit of Toxic Waste from Smelter Factory Processing Pyrite Raw Materials.

Saprotrophic microfungi associated with roots of Calamagrostis epigeios growing in industrial waste contaminated with high concentrations of heavy metals (manganese, iron, zinc) were isolated on three different media. The isolates were obtained from three plots colonized by plant cenoses, representing three different succession series of plant cover: initial, intermediate, and advanced. A total of 60 species of saprotrophic microfungi were found in the roots during three samplings. In addition, unidentified dark and white sterile mycelia were frequently isolated. Trichoderma koningii was frequent in the roots formed under the advanced phytocenose. The abundance of dark sterile mycelium gradually decreased with progressing succession of the plant cover. Chrysosporium merdarium was rarely observed in the roots under the intermediate and advanced plant cenoses. Multivariate redundancy analysis showed that the effect of succession stage of phytocenoses on incidence of saprotrophic microfungi in mycocenoses present in roots was statistically significant. When the similarity of root mycocenoses of the three plots was studied using the similarity coefficients, the results proved that the similarities between the initial and intermediate or intermediate and advanced mycocenoses were higher than that between the initial and advanced mycocenoses. This corresponds to a different speed of succession of the mycocenoses. The results suggest that the dark sterile mycelia, abundant at slowly developing plots, play a dominant role during the first stages of succession where the plant cover is poor.

Journal Article↗

PubMed1998

Oat beta-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains.

Novel oligomers that resist digestion in the upper gut were prepared from oat mixed-linked beta-glucan and xylan by enzymatic hydrolysis with lichenase of Bacillus subtilis and xylanase of Trichoderma reesei respectively. The low-molecular-mass hydrolysis products of beta-glucan and xylan were compared with fructooligomers and raffinose in their ability to provide growth substrates for probiotic (Lactobacillus and Bifidobacterium) and intestinal (Bacteroides, Clostridium and Escherichia coli) strains in vitro. A degradation profile of each carbohydrate and total sugar consumption were analysed with HPLC, and bacterial growth rate with an automatic turbidometer, the Bioscreen C system. beta-Glucooligomers and xylooligomers both enhanced the growth of health-promoting probiotic strains as compared with intestinal bacterial growth, but not to a significant level. Raffinose stimulated the probiotic strains significantly, whereas fructooligomers induced high average growth for intestinal bacteria also.

Bifidobacterium↗

PubMed1999

Processes of liquefaction/solubilization of Spanish coals by microorganisms

Several fundamental aspects of microbial coal liquefaction/solubilization were studied. The liquefied/solubilized products from coal by microorganisms were analysed. The liquid products analysed by IR titration and UV/visible spectrometry showed some alterations with regard to the original coal. Humic acids extracted from the liquefied lignite showed a reduction in the average molecular weight and a increase in the condensation index, probably due to depolymerization caused by microorganisms. The mechanisms implicated in coal biosolubilization by two fungal strains, M2 (Trichoderma sp.) and M4 (Penicillium sp.) were also studied. Extracellular peroxidase, esterase and phenoloxidase enzymes appear to be involved in coal solubilization.

Journal Article↗

PubMed1999

Biosynthesis of polyhydroxyalkanoates from low-rank coal liquefaction products by Pseudomonas oleovorans and Rhodococcus ruber

A screening identified several bacteria that were able to use chemically heterogeneous low-rank coal liquefaction products as complex carbon sources for growth. Pseudomonas oleovorans and Rhodococcus ruber accumulated polyhydroxyalkanoic acids (PHA) amounting to 2%-8% of the cell dry weight when the cells were cultivated on these liquefaction products in the absence of any other carbon source. R. ruber accumulated, in addition to PHA, small amounts of triacylglycerols. The accumulated PHA consisted of 3-hydroxyhexanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate (P. oleovorans) or 3-hydroxybutyric acid and 3-hydroxyvaleric acid (R. ruber). Low-rank coal liquefaction products obtained from Trichoderma atroviride were better substrates for P. oleovorans than chemically produced fulvic acids.

Journal Article↗

PubMed2003

Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent.

Phylogenetic characterization of soil isolate NJ-15, based on sequence homology of a partial 746-bp fragment of 16SrDNA amplicon, with the ribosomal database sequences (http://www.msu.edu/RDP/cgis/phylip.cgi), validated the strain as Pseudomonas aeruginosa. The strain NJ-15 produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. Besides, the strain also exhibited significant production of both the siderophore and hydrogen cyanide (HCN) on chrome azurol S and King's B media, respectively. The data revealed lower HCN production under iron-limiting conditions vis-à-vis higher HCN release with iron stimulation. Significant growth inhibition of phytopathogenic fungi occurred in the order as Fusarium oxysporum > Trichoderma herizum > Alternaria alternata > Macrophomina phasiolina upon incubation with strain NJ-15 cells. Thus, the secondary metabolites producing new Pseudomonas aeruginosa strain NJ-15 exhibited innate potential of plant growth promotion and biocontrol activities in vitro.

Antibiosis↗

PubMed2005

Sequential gene deletions in Hypocrea jecorina using a single blaster cassette.

In Hypocrea jecorina (anamorph: Trichoderma reesei) multiple gene deletions are limited by the number of readily available selection markers. We have therefore constructed a blaster cassette which enables successive gene knock-outs in H. jecorina. This 3.5 kb pyr4 blaster cassette contains the H. jecorina pyr4 marker gene encoding orotidine-5'-monophosphate (OMP) decarboxylase flanked by two direct repeats of the Streptoalloteichus hindustanus bleomycin gene (Sh ble), which facilitate the excision of the blaster cassette by homologous recombination after each round of deletion. Functionality of this pyr4 blaster cassette was demonstrated by deletion of the glk1 encoding glucokinase and hxk1 encoding hexokinase. 1.4-1.8 kb of the non-coding flanking regions of both target genes were cloned into the respective blaster cassettes and transformation of a pyr4 negative H. jecorina strain with the two cassettes resulted in 10-13% of the transformants in the deletion of one of the two kinase genes. For excision of the pyr4 blaster cassettes, Deltaglk1 strains were selected for growth in the presence of 5-fluoroorotic acid. Recombination between the two Sh ble elements resulted in uridine auxotrophic strains which retained their respective glucokinase negative phenotype. Subsequent transformation of one of these auxotrophic Deltaglk1 strains with the hexokinase blaster cassette resulted in pyr4 prototrophic strains deleted in both glk1 and hxk1. Deltaglk1 strains showed reduced growth on d-glucose and d-fructose whereas Deltahxkl strains showed reduced compact growth on d-glucose but were unable to grow on d-fructose as carbon source. The double Deltaglk1Deltahxk1 deletion strain was completely unable to grow on either d-glucose or d-fructose.

Amino Acid Sequence↗

PubMed2000

Glucose dependent transcriptional expression of the cre1 gene in Acremonium chrysogenum strains showing different levels of cephalosporin C production.

The cre1 gene from the beta-lactam producer Acremonium chrysogenum has been isolated and characterized in order to study glucose-dependent gene expression in this biotechnically important fungus. The deduced protein sequence is highly similar to amino-acid sequences of other known glucose repressors from filamentous fungi, and carries conserved zinc-finger and regulatory motifs. Contrary to cre gene expression in Trichoderma reesei and Aspergillus nidulans, the transcript level of the cre1 gene from an A. chrysogenum wild-type strain is increased in the presence of glucose. Remarkably, the glucose-dependent transcriptional upregulation does not take place in another A. chysogenum strain, which displays enhanced production of the beta-lactam antibiotic cephalosporin C. We surmise that the de-regulation of cre1 is connected with the increased production rate in this strain.

Acremonium↗

PubMed1997

Identification of genes induced in alkaloid-producing cultures of Claviceps sp.

In order to identify genes which are expressed during alkaloid synthesis in an axenic culture of Claviceps sp. (strain ATCC 26245), a cDNA library from a producing culture was differentially screened with cDNA from producing (cDNA+) and non-producing (cDNA-) cultures, respectively. Altogether, ten cDNA clones were obtained, the alkaloid-synthesis-correlated expression of which was confirmed by Northern analyses. Evaluation of their nucleotide and derived amino-acid sequences identified one gene unequivocally, coding for dimethylallyltryptophan-synthase (DMAT-S), the initial enzyme of the specific alkaloid pathway. For two other genes significant homologies to known fungal genes were detected: one clone showed homology to the Neurospora crassa ccg1 gene, coding for a clock-regulated putative general stress protein; seven cDNA clones, derived from the same gene, which is highly expressed under these conditions, contained typical hydrophobin domains and long stretches of asparagine/glycine repeats (like QID3 from Trichoderma harzianum), thus probably representing a cell-wall constituent. These data show that this is not only a successful approach to clone genes specific for the alkaloid-pathway of C. purpurea, but also of genes which might be involved in the differentiation of sclerotial hyphae, the prerequisite for alkaloid synthesis.

Alkaloids↗

PubMed2002

Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity.

Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity.

Catalysis↗

PubMed2004

An agglutinating chitinase with two chitin-binding domains confers fungal protection in transgenic potato.

Brassica juncea BjCHI1 is a unique chitinase with two chitin-binding domains. Here, we show that, unlike other chitinases, potato-expressed BjCHI1 shows hemagglutination ability. BjCHI1 expression in B. juncea seedlings is induced by Rhizoctonia solani infection, suggesting its protective role against this fungus. To verify this, transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing BjCHI1 generated by Agrobacterium-mediated transformation were challenged with R. solani. We also transformed potato with a cDNA encoding Hevea brasiliensis beta-1,3-glucanase, designated HbGLU, and a pBI121-derivative that contains cDNAs encoding both BjCHI1 and HbGLU. In vitro fungal bioassays using Trichoderma viride showed that extracts from transgenic potato lines co-expressing BjCHI1 and HbGLU inhibited fungal growth better than extracts from transgenic potato expressing either BjCHI1 or HbGLU, suggesting a synergistic effect. Consistently, in vivo fungal bioassays with soil-borne R. solani on young transgenic potato plants indicated that the co-expressing plants showed healthier root development than untransformed plants or those that expressed either BjCHI1 or HbGLU. Light microscopy and transmission electron microscopy revealed abundant intact R. solani hyphae and monilioid cells in untransformed roots and disintegrated fungus in the BjCHI1-expressing and the BjCHI1 and HbGLU co-expressing plants. Observations of collapsed epidermal cells in the co-expressing potato roots suggest that these proteins effectively degrade the fungal cell wall, producing elicitors that initiate other defense responses causing epidermal cell collapse that ultimately restricts further fungal penetration.

Amino Acid Sequence↗

PubMed1999

Cloning of the cbhI and cbhII genes involved in cellulose utilisation by the straw mushroom Volvariella volvacea.

The straw mushroom Volvariella volvacea is cultivated on substrates rich in cellulose and has been shown to produce a family of cellulolytic enzymes. A PCR-based strategy was adopted to clone genes involved in cellulose utilisation, using degenerate primers designed to amplify conserved catalytic domain sequences of cellobiohydrolases (CBHs). PCR with these primers produced two DNA fragments with sequence similarity to the cbhI and cbhII gene families detected in Trichoderma, Phanerochaete and Agaricus species. Full-length clones of these genes were obtained from an EMBL3 genomic library, and RACE-PCR was used to verify the presence of introns. The cbhI homologue has a coding region of 1722 bp, containing two introns, generating a 536 amino acid polypeptide product. The cbhII gene has a coding region of 1693 bp, containing five introns, and gives rise to a 470-amino acid polypeptide product. Northern and PCR analyses were used to study the expression of the genes. These revealed that transcripts of both genes were induced on medium containing cellulose with cbhI being expressed more strongly than cbhII - but were repressed on medium containing glucose.

Agaricales↗

PubMed2004

Arbuscular mycorrhiza and Collembola interact in affecting community composition of saprotrophic microfungi.

The functioning of the plant-mycorrhiza system depends on interactions with other organisms, including saprotrophic (ST) soil fungi. The interactions between mycorrhizal and ST fungi are likely affected by fungivorous soil animals, such as Collembola. In a two-factorial laboratory experiment lasting for 30 weeks we assessed the effects of an arbuscular mycorrhizal fungus (Glomus mosseae) and Collembola (Protaphorura fimata, Heteromurus nitidus and Folsomia candida) on the community composition of ST microfungi in soil planted with the invasive grass Cynodon dactylon. The presence of mycorrhiza substantially reduced total plant biomass and reduced N and P availability to the soil microflora, though these effects were less pronounced in the presence of Collembola. The density of Collembola was high (corresponding to about 2x10(5) individuals m-2) and was not affected by the presence of G. mosseae. In spite of the large amount of mycorrhizal mycelium in soil, it contributed little to Collembola nutrition. The presence of mycorrhiza strongly affected the community structure of ST soil fungi. In particular, mycorrhiza reduced the relative abundance of Trichoderma harzianum and Exophiala sp., but increased the abundance of Ramichloridium schulzeri and several sterile forms. However, the difference between fungal communities in mycorrhizal and non-mycorrhizal treatments was much more pronounced in the presence of Collembola. Presumably, the intense grazing by Collembola destabilized the ST fungal community, thereby making it more susceptible to the influence of G. mosseae. These results document for the first time that fungal feeding soil invertebrates can significantly affect the interactions between mycorrhizal fungi and ST soil microorganisms.

Animals↗

PubMed1998

Fungal peritonitis in patients on continuous ambulatory peritoneal dialysis.

The purpose of this study was to analyze the microbiological and clinical features of fungal peritonitis in patients with endstage renal failure treated with continuous ambulatory peritoneal dialysis (CAPD). The diagnosis of peritonitis was based on abdominal discomfort or pain, cloudy peritoneal effluent with an elevated leukocyte count and isolation of fungi from the peritoneal effluent. Amphotericin B, flucytosine, ketoconazole, miconazole and more recently fluconazole were used for antifungal therapy. From 1983 to 1997 13 patients experienced 14 episodes of fungal peritonitis, comprising 3.1% of all episodes of peritonitis in the dialysis centre. Isolates from the peritoneal effluent comprised Candida tropicalis in two cases, Candida parapsilosis in two cases, Candida albicans in one case, Candida lusitaniae in one case,Cephalosporium spp. in three cases, Aspergillus fumigatus in two cases, and an Aspergillus sp., a Trichoderma sp. and a yeast in one case each. In eight cases bacterial infection shortly before the episode of fungal peritonitis was documented. In 12 (86%) cases the peritoneal catheter had to be removed. Four patients died during the treatment, and one patient died 2 months after the end of treatment due to intra-abdominal bleeding from peritoneal adhesions. Only two patients continued CAPD later; the other patients were switched to hemodialysis. It is concluded that fungal peritonitis is a rare but serious complication in CAPD patients with high rates of morbidity, mortality and drop-out from the CAPD programme (85%). The most frequent isolates were Candida spp. A predisposing factor for fungal peritonitis could be a recent bacterial infection treated with antibiotics. Early peritoneal catheter removal is recommended.

Adult↗

PubMed2005

Degradation of cellulose by the bean-pathogenic fungus Colletotrichum lindemuthianum. Production of extracellular cellulolytic enzymes by cellulose induction.

Colletotrichum lindemuthianum was able to grow and produce extracellular cellulolytic activity in a defined medium containing cellulose as the main carbon substrate. As measured either by the hydrolysis of 4-methylumbelliferyl-beta-D -cellotrioside or the release of glucose from carboxymethylcellulose, activity reached a peak after 13 days of incubation and then declined whereas growth markedly increased afterwards. Detection of glucose in carboxymethylcellulose hydrolysates suggested the concerted operation of endo-1,4-beta-glucanase, cellobiohydrolase (exo-1,4-beta-glucanase) and beta-glucosidase activities. The highest levels of cellulolytic activity were obtained in media supplemented with cellulose and glutamate. Other carbon and nitrogen sources markedly influenced growth and enzyme production. Oligonucleotides homologous to specific regions of the cellobiohydrolase-encoding cbhII gene from Trichoderma reesei were used to isolate a C. lindemuthianum cbhII-DNA fragment whose sequence revealed homologies of 98% and 92% with the nucleotide and the deduced amino acid sequences of the corresponding cbhII-DNA of T. reesei, respectively. RT-PCR and Southern blot analyses of total RNA samples obtained from cellulose-grown but not from glucose-grown mycelium revealed the expression of the corresponding cbhII transcript. The cbhII-cDNA fragment was cloned and sequenced.

Amino Acid Sequence↗

PubMed2004

Function of a low molecular peptide generated by cellulolytic fungi for the degradation of native cellulose.

Peptides (MW < 5 kDa) produced by 57 cellulolytic fungi can form free radicals. Scanning tunneling microscopy (STM) and IR spectroscopy showed that the peptide produced by Trichoderma pseudokoningii can break the hydrogen bond network of cellulose. The synergic action of these peptides and cellulases increased production of reducing sugars during degradation of native cellulose.

Biodegradation, Environmental↗

PubMed2005

Expression of a self-processing, pathogen resistance-enhancing gene construct in Arabidopsis.

A gene cassette, p35S-CNO, was designed to express three gene products driven by a single constitutive CaMV 35S promoter. The individual coding regions were linked in frame to produce a single polyprotein, using spacer sequences encoding a specific heptapeptide cleavage recognition site (ENLYFQS) for the nuclear-inclusion-a (NIa) proteinase of tobacco etch virus (TEV). The protein coding sequences used were: a Trichoderma harzinum endochitinase, a truncated NIa proteinase of TEV, and a wheat oxalate oxidase. When p35S-CNO construct was tested in Arabidopsis thaliana, the polyprotein was properly cleaved after translation and the products exhibited functional enzymatic activity in vivo.

Arabidopsis↗