Zsuzsanna Antal

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

BackgroundMycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.ResultsHere we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.ConclusionsThe data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Green Mold Diseases of Agaricus and Pleurotus spp. Are Caused by Related but Phylogenetically Different Trichoderma Species

ABSTRACT Producers of champignon (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus) are facing recent incidents of green mold epidemics in Hungary. We examined 66 Trichoderma strains isolated from Agaricus compost and Pleurotus substrate samples from three Hungarian mushroom producing companies by a polymerase chain reaction-based diagnostic test for T. aggressivum, sequence analysis of the internal transcribed spacer region 1 (ITS1) and ITS2 and (selectively) of the fourth and fifth intron of translation elongation factor 1alpha (tef1alpha), and restriction fragment length polymorphism of mitochondrial DNA. Seven Trichoderma species were identified: T. aggressivum f. europaeum (17 isolates), T. harzianum (three isolates), T. longibrachiatum (four isolates), T. ghanense (one isolate), T. asperellum (four isolates), T. atroviride (nine isolates), and a still undescribed phylogenetic species, Trichoderma sp. DAOM 175924 (28 isolates). T. aggressivum f. europaeum was exclusively derived from A. bisporus compost, whereas Trichoderma sp. DAOM 175924 exclusively occurred in the substrate for Pleurotus cultivation. Sequences of the latter strains were co-specific with those for Trichoderma pathogens of P. ostreatus in Korea. The widespread occurrence of this new species raises questions as to why infections by it have just only recently been observed. Our data document that (i) green mold disease by T. aggressivum f. europaeum has geographically expanded to Central Europe; (ii) the green mold disease of P. ostreatus in Hungary is due to the same Trichoderma species as in Korea and the worldwide distribution of the new species indicates the possibility of spreading epidemics; and (iii) on mushroom farms, the two species are specialized on their different substrates.

Alternative reproductive strategies of Hypocrea orientalis and genetically close but clonal Trichoderma longibrachiatum, both capable of causing invasive mycoses of humans.

The common soil fungus Trichoderma (teleomorph Hypocrea, Ascomycota) shows increasing medical importance as an opportunistic human pathogen, particularly in immunocompromised and immunosuppressed patients. Regardless of the disease type and the therapy used, the prognosis for Trichoderma infection is usually poor. Trichoderma longibrachiatum has been identified as the causal agent in the majority of reported Trichoderma mycoses. As T. longibrachiatum is very common in environmental samples from all over the world, the relationship between its clinical and wild strains remains unclear. Here we performed a multilocus (ITS1 and 2, tef1, cal1 and chit18-5) phylogenetic analysis of all available clinical isolates (15) and 36 wild-type strains of the fungus including several cultures of its putative teleomorph Hypocrea orientalis. The concordance of gene genealogies recognized T. longibrachiatum and H. orientalis to be different phylogenetic species, which are reproductively isolated from each other. The majority of clinical strains (12) were attributed to T. longibrachiatum but three isolates belonged to H. orientalis, which broadens the phylogenetic span of human opportunists in the genus. Despite their genetic isolation, T. longibrachiatum and H. orientalis were shown to be cosmopolitan sympatric species with no bias towards certain geographical locations. The analysis of haplotype association, incongruence of tree topologies and the split decomposition method supported the conclusion that H. orientalis is sexually recombining whereas strict clonality prevails in T. longibrachiatum. This is a rare case of occurrence of sexual reproduction in opportunistic pathogenic fungi. The discovery of the different reproduction strategies in these two closely related species is medically relevant because it is likely that they would also differ in virulence and/or drug resistance. Genetic identity of environmental and clinical isolates of T. longibrachiatum and H. orientalis suggests the danger of nosocomial infections by Hypocrea/Trichoderma and highlights the need for ecological studies of spore dispersal as source of invasive human mycoses.

Infectious keratitis caused by Aspergillus tubingensis.

Purpose: To report 2 cases of keratomycosis caused by Aspergillus tubingensis. Methods: The therapeutic courses were recorded for 2 male patients, 52 and 78 years old, with fungal keratitis caused by black Aspergillus strains. Morphological examination of the isolates was carried out on malt extract agar plates. A segment of the β-tubulin gene was used for molecular identification. Antifungal susceptibilities were determined by the E test method for molds and the broth microdilution technique National Committee for Clinical Laboratory Standards M38-A. Results: A 52-year-old man presented with complaints of pain and redness in the right eye. The patient was successfully treated with natamycin and econazole eyedrops, itraconazole eye ointment, and oral ketoconazole. A 78-year-old man presented with total corneal necrosis in the right eye. A therapeutic keratoplasty was performed, and topical natamycin and econazole were applied. At the postoperative visit after 3 weeks, almost the full corneal graft was clear with formed anterior chamber. Black Aspergillus strains were isolated from the corneal scrapings of both cases and initially identified as Aspergillus niger based on culture characteristics. Sequence analysis of a segment of the β-tubulin gene revealed that the isolates are representatives of A. tubingensis. Conclusions: Aspergillus tubingensis is closely related with A. niger, the differentiation of these 2 species is difficult by classical morphological criteria. To our knowledge, the presented cases of fungal keratitis are the first reports on ocular infection caused by A. tubingensis.

A method for high-frequency transformation of Trichoderma viride

Abstract A heterologue plasmid vector-mediated transformation system has been developed for the filamentous fungus Trichoderma viride . Transformants were obtained by using the plasmid pCSN43 carrying the Escherichia coli hygromycin B resistance gene under the control of Aspergillus nidulans trpC promoter and terminator. The transformation frequency was more than 10 4 μg −1 plasmid DNA when nuclease inhibitors were used. Hybridization experiments revealed the presence of replicative plasmids of different sizes in the fungal genome.

Molecular diagnosis, epidemiology and taxonomy of emerging medically important filamentous fungi

Relatively uncommon, but emerging filamentous fungal pathogens are of increasing clinical importance, mainly as the causative agents of opportunistic infections in the expanding population of immunocompromised patients. The classical diagnostic, typing and taxonomic methods for the investigation of some filamentous fungi have their limitations. However, rapidly developing molecular biological methods may provide the clinical microbiologist with valuable help. Molecular techniques with the potential for successful use in the clinical diagnostics of these fungi involve methods based on species-specific primers, in situ hybridization, RFLP of 18S rDNA, and sequence analysis of rDNA and/or ITS regions. PCR fingerprinting, mtDNA and genomic RFLP, RAPD, PFGE, sequence analysis of the ITS region and analysis of microsatellite sequences have been successfully used for their genotyping producing valuable data on their epidemiology. Molecular techniques have also proved to be very helpful by providing important new taxonomic information on these fungi which may facilitate the development of species-specific molecular diagnostic tools. The successes achieved in the publications reviewed suggest that molecular methods for the diagnosis and epidemiological investigations of these fungal pathogens should (and hopefully will) become routinely applied tools in clinical microbiology laboratories.

Black Aspergilli in tropical infections

Members of the genus Aspergillus are among the filamentous fungal agents frequently causing infections in humans. A. fumigatus is the most commonly isolated fungal pathogen within the genus; however, other species, including A. terreus, A. flavus and A. niger are also of increasing importance. Infections caused by black aspergilli are especially frequent in developing countries with hot, humid, tropical or semitropical climates. Several reports revealed A. niger as the most common causative agent of otomycosis and fungal keratitis in certain geographic regions. Species related to A. niger are difficult to identify on the basis of morphological criteria alone. Recent molecular analyses revealed that apart from A. niger, other black aspergilli, including A. tubingensis and the recently described species A. brasiliensis are also potential causative agents of human infections. In the case of corneal infections, the available data indicate that these species may be responsible for a significant proportion of cases caused by black aspergilli. The aim of this review is to summarize the literature data about the involvement of black aspergilli (members of Aspergillus section Nigri) in human infections under tropical and semitropical climates, including otomycosis and eye infections. Available retrospective studies are summarized with special emphasis on the clinical manifestation, epidemiological aspects and therapeutical possibilities; furthermore, the molecular methods for the exact diagnosis and identification of black Aspergillus strains are also discussed.

Transformation of a mycoparasitic Trichoderma harzianum strain with the argB gene of Aspergillus nidulans

A transformation system was developed for the mycoparasitic filamentous fungus Trichoderma harzianum, based upon complementation of auxotrophic mutants. Prototrophic transformants were obtained using plasmids pSal23 and pAN5-41B, carrying the Aspergillus nidulans argB gene, and both the Aspergillus nidulans argB and Escherichia coli lacZ genes, respectively.

PURIFICATION AND PRELIMINARY CHARACTERIZATION OF A COLD-ADAPTED EXTRACELLULAR PROTEINASE FROM TRICHODERMA ATROVIRIDE

Eleven cold-tolerant Trichoderma isolates were screened for the production of proteolytic activities at 10 degrees C. Based on the activity profiles determined with paranitroanilide substrates at 5 degrees C, strain T221 identified as Trichoderma atroviride was selected for further investigations. The culture broth of the strain grown at 10 degrees C in casein-containing culture medium was concentrated by lyophilization and subjected to gel filtration, which was followed by chromatofocusing of the fraction showing the highest activity on N-benzoyl-Phe-Val-Arg-paranitroanilide. The purified enzyme had a molecular weight of 24 kDa, an isoelectric point of 7.3 and a pH optimum of 6.2. The temperature optimum of 25 degrees C and the low thermal stability suggested that it is a true cold-adapted enzyme. Substrate specificity data indicate that the enzyme is a proteinase with a preference for Arg or Lys at the P1 position. The effect of proteinase inhibitors suggests that the enzyme has a binding pocket similar to the one present in trypsin.

Isolation a new carbendazim degrading bacterium from Hungarian agricultural soil samples and its preliminary characterization

One of the biggest challenges for the safe maize production is the yield stability in a wide range of environments with different soil fertility, weather conditions, prevailing pests and diseases as well as cultural practices. In order to achieve it, new hybrids with higher tolerance to biotic and abiotic stress are continuously being created. In order to compare performance of maize hybrids in environments with different levels and types of stress, and relate it to the stalk lodging incidence, we compared performance of 64 maize hybrids obtained by crossing 16 inbred lines with four inbred testers in three different environments. Two environments at same location (Rugvica) differed in crop rotation, and the third environment at location Botinec was considered as dry because of low water capacity of the soil. Mixed models that included all design elements and genetic background of hybrids were used to analyze the effect of lodging on yield in different environments. The results show that stalk lodging had a significant effect on yield, but significant lodging x environment interaction indicates that this effect was rather environment specific.Sweet pepper Istra F1 hybrid was grown in a greenhouse on K rich soil. For evaluation of K antagonism, two K rates (55 and 85 kg ha-1) were applied in a randomized complete block design with four replications. Total number of fruits, total number of BER affected fruits as well as average fruit mass were recorded for each harvest. Fruit and leaf K and Ca content were determined at three plant growth stages (at the stage of the first, third and fifth fruit cluster). Higher K rate gave higher : average fruit mass (169.45 g), total number of fruits per plant (7.95), number of BER affected fruits per plant (3.82), K in leaves (5.44% DM), K in fruits (6.35% DM), but negatively correlated with Ca concentration in fruits (0.57% DM) and leaves (3.24% DM). This resulted in decreased marketable yield of sweet pepper fruits.