Identification of loci and functional characterization of trichothecene biosynthesis genes in filamentous fungi of the genus Trichoderma.

Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function.

Biodiversity of Trichoderma strains in Tunisia.

Trichoderma strains were sampled in 4 different bioclimatic zones of Tunisia, a Mediterranean North African country with strong climatic and edaphic variability from north to south, to assess the genetic diversity of endemic species of Trichoderma and their relationship to the bioclimatic zones. In all, 53 strains were isolated and identified at the species level by analysis of their internal transcribed spacers regions 1 and 2 (ITS1 and ITS2) of the rDNA cluster and (or) a fragment of the translation elongation factor 1 (tef1) gene, using an online interactive key for species identification in Trichoderma and ex-type strains and taxonomically established isolates of Trichoderma as references. At least 2 different species were observed in each ecosystem. Trichoderma harzianum clade VI and Trichoderma longibrachiatum were present in forest soils in north Tunisia; Trichoderma atroviride and Trichoderma hamatum were found in cultivated fields in northeast Tunisia; T. harzianum clade VI, a Trichoderma sp. close to the T. harzianum complex, and Trichoderma saturnisporum were isolated from forest soils in central Tunisia; and T. harzianum clade II and T. hamatum were present in oasis soils in south Tunisia.

[Clinical experience with efalizumab in the Hospital of Cruces]. / Experiencia clínica con efalizumab en el Hospital de Cruces.

Efalizumab (Raptiva) is one of the biological agents approved recently for the treatment of patients with moderate-severe psoriasis who have not responded to conventional treatments. It is a humanized IgG1 monoclonal antibody which, due to its anti-D11 effect, is capable of blocking the endothelial migration and T cell activation on the skin, fundamental processes in the etiopathogeny of psoriasis. We present the experience we have had in our hospital over the last two years with 23 patients who have initiated treatment with efalizumab.

Cloning and characterization of the erg1 gene of Trichoderma harzianum: effect of the erg1 silencing on ergosterol biosynthesis and resistance to terbinafine.

Trichoderma species are commonly used as biocontrol agents of different plant-pathogenic fungi. Terpene compounds are involved in the biocontrol process due to their antifungal properties (e.g., ergokonins and viridins) but additionally their structural function in the cell membranes (ergosterol) is essential. We report here the characterization of the T. harzianum erg1 gene, encoding a squalene epoxidase, a key enzyme in the biosynthesis of triterpene derivatives such as ergosterol. In T. harzianum the partial silencing of the erg1 gene gave rise to transformants with a higher level of sensitivity to terbinafine, an antifungal compound that acts specifically over the squalene epoxidase activity. In addition, these silenced transformants produced lower levels of ergosterol than the wild type strain. Finally, the silencing of the erg1 gene resulted in an increase in the expression level of the erg7 gene that encodes the oxidosqualene lanosterol-cyclase, another enzyme of the terpene biosynthesis pathway.

Specific PCR assays for the detection and quantification of DNA from the biocontrol strain Trichoderma harzianum 2413 in soil.

Strain identification in situ is an important factor in the monitoring of microorganisms used in the field. In this study, we demonstrated the use of sequence-characterized amplified region (SCAR) markers to detect genomic DNA from Trichoderma harzianum 2413 from soil. Two primers (SCAR A1/SCAR A1c) were tested against DNA of 27 isolates of Trichoderma spp. and amplified a 990-bp fragment from T. atroviride 11 and a 1.5-kb fragment from T. harzianum 2413, using an annealing temperature of 68 degrees C. These fragments showed no significant homology to any sequence deposited in the databases. The primer pair, BR1 and BR2, was designed to the 1.5-kb fragment amplified from T. harzianum 2413, generating a SCAR marker. To test the specificity of these primers, experiments were conducted using the DNA from 27 Trichoderma spp. strains and 22 field soil samples obtained from four different countries. PCR results showed that BR1 and BR2 amplified an 837-bp fragment unique to T. harzianum 2413. Assays in which total DNA was extracted from sterile and nonsterile soil samples, inoculated with spore or mycelium combinations of Trichoderma spp. strains, indicated that the BR1 and BR2 primers could specifically detect T. harzianum 2413 in a pool of mixed DNA. No other soil-microorganisms containing these sequences were amplified using these primers. To test whether the 837-bp SCAR marker of T. harzianum 2413 could be used in real-time PCR experiments, new primers (Q2413f and Q2413r) conjugated with a TaqMan fluorogenic probe were designed. Real-time PCR assays were applied using DNA from sterile and nonsterile soil samples inoculated with a known quantity of spores of Trichoderma spp. strains.

N-benzoyl-N'-alkylthioureas and their complexes with Ni(II), Co(III) and Pt(II) - crystal structure of 3-benzoyl-1-butyl-1-methyl-thiourea: activity against fungi and yeast.

The thiourea derivatives of N-butylmethylamine (3-benzoyl-1-butyl-1-methyl-thiourea) (1), N-ethylisopropylamine (3-benzoyl-1-ethyl-1-isopropyl-thiourea) (2) and the corresponding complexes of 1 and 2 with Ni(II), Co(III) and Pt(II) have been synthesized. The compounds obtained were characterized by elemental analysis, spectroscopic methods (FT-IR, UV-Vis and NMR) and mass spectrometry. Compound 1, crystallized in the triclinic space group. The antifungal activities of compounds 1 and 2 and their corresponding complexes against the fungus Penicillium digitatum and against the yeast Saccharomyces cerevisiae were investigated. In general, fungal growth inhibition was higher with compound 1 and its complexes than with compound 2, except for the Co(III) complex of 2.

Development of a strain-specific SCAR marker for the detection of Trichoderma atroviride 11, a biological control agent against soilborne fungal plant pathogens.

The genus Trichoderma includes biocontrol agents (BCAs) effective against soilborne plant pathogenic fungi. Several potentially useful strains for biological control are difficult to distinguish from other strains of Trichoderma found in the field. So, there is a need to find ways to monitor these strains when applied to natural pathosystems. We have used random amplified polymorphic DNA (RAPD) markers to estimate genetic variation among sixteen strains of the species T. asperellum, T. atroviride, T. harzianum, T. inhamatum and T. longibrachiatum previously selected as BCAs, and to obtain fingerprinting patterns. Analysis of these polymorphisms revealed four distinct groups, in agreement with previous studies. Some of the RAPD products generated were used to design specific primers. Diagnostic PCR performed using these primers specifically identify the strain T. atroviride 11, showing that DNA markers may be successfully used for identification purposes. This SCAR (sequence-characterised amplified region) marker can clearly distinguish strain 11 from other closely related Trichoderma strains.

Molecular characterization and identification of biocontrol isolates of Trichoderma spp.

The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as "Trichoderma harzianum" Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.

Chloride and ethyl ester morpholine thiourea derivatives and their Ni(II) complexes. Crystal and molecular structures of the thiourea derivative L-leucine methyl ester and its complexes with Cu(II) and Pt(II). Growth of the pathogenic fungus Botrytis cinerea.

We have synthesized a series of ligands (1, 3, 4, 6 and 7) and some of their complexes with Ni(II), Cu(II) and Pt(II) (2, 5, 8 and 9). These compounds were studied and characterized by elemental analysis, IR and UV-Vis spectra, conductivity measurements in solution, FAB+/MS, 1H and 13C NMR, ESR, etc. Compound 7 crystallized in the orthorhombic space group P2(1)2(1)2(1), with Z = 4. Unit cell parameters were as follows: a = 21.307(2) A, alpha = 90 degrees, b = 12.498(1) A, beta = 90 degrees, c = 7.7232(4) A, gamma = 90 degrees. For seven of these compounds, the antifungal activity of a major pathogen responsible for important crop damage was studied. In general, inhibition by the ligands was higher than that of the complexes. When the thiourea was linked to some diethyl groups, the compounds showed higher antifungal activity than the morpholine groups. Compound 3 achieved total inhibition (100%).

Isolation of Trichoderma harzianum Th2 from Commercial Mushroom Compost in Spain.

Trichoderma spp. are associated with green mold of mushrooms. This fungal disease has caused severe losses in mushroom production in countries such as Ireland, the United Kingdom, Canada, and the United States. This disease is caused by two biotypes of T. harzianum (Th2, Europe; Th4, North America) (1,2). Both biotypes have not been detected in mushrooms or other material in Spain previously. However, during 1998, green mold was detected at facilities dedicated to produce compost, as well as in facilities used to produce Agaricus bisporus (Lange) Imbach. Three compost samples were isolated from commercial bags with mushroom substrate and three more samples were taken from mushroom yards. Several spores were isolated by the dilution plate method. Initial identification of the pathogenic fungi was made by examining cultures grown on potato dextrose agar. Morphological characteristics of all isolates coincided with the description of T. harzianum (3). The following amounts of CFU per g were found in commercial compost samples: 1.2 × 108, 5.5 × 107, and 1.4 × 107 per g; whereas 3 × 108, 12.4 × 107, and 2.2 × 106 were obtained from mushroom yards. The fragment containing the internal transcribed spacer (ITS1) was amplified and sequenced for each of the six samples obtained. The ITS1 sequence (201 bp) was identical in all samples, and the sequence was aligned, with Clustal W, with Th2 and Th4 biotype sequences of the EMBL data base. The ITS1 sequence showed 0.55% divergence from Th2 isolates and more distance, 3.3%, with Th4 isolates. The ITS1 sequence obtained with all Spanish samples studied, EMBL accession number AJ1321550, was identical to that described for the Irish isolate Th2I (#63), with accession number U78880 in the EMBL data base (1). This is the first description of the Th2 biotype in Spain. References: (1) M. D. Ospina-Giraldo et al. Mycologia 90:76, 1998. (2) D. L. Rinker et al. Mushroom World 8:71, 1997. (3) D. A. Seaby. Plant Pathol. 45:905, 1996.