The worldwide commercial production of the oyster mushroom Pleurotus ostreatus is currently threatened by massive attacks of green mold disease. Using an integrated approach to species recognition comprising analyses of morphological and physiological characters and application of the genealogical concordance of multiple phylogenetic markers (internal transcribed spacer 1 [ITS1] and ITS2 sequences; partial sequences of tef1 and chi18-5), we determined that the causal agents of this disease were two genetically closely related, but phenotypically strongly different, species of Trichoderma, which have been recently described as Trichoderma pleurotum and Trichoderma pleuroticola. They belong to the Harzianum clade of Hypocrea/Trichoderma which also includes Trichoderma aggressivum, the causative agent of green mold disease of Agaricus. Both species have been found on cultivated Pleurotus and its substratum in Europe, Iran, and South Korea, but T. pleuroticola has also been isolated from soil and wood in Canada, the United States, Europe, Iran, and New Zealand. T. pleuroticola displays pachybasium-like morphological characteristics typical of its neighbors in the Harzianum clade, whereas T. pleurotum is characterized by a gliocladium-like conidiophore morphology which is uncharacteristic of the Harzianum clade. Phenotype MicroArrays revealed the generally impaired growth of T. pleurotum on numerous carbon sources readily assimilated by T. pleuroticola and T. aggressivum. In contrast, the Phenotype MicroArray profile of T. pleuroticola is very similar to that of T. aggressivum, which is suggestive of a close genetic relationship. In vitro confrontation reactions with Agaricus bisporus revealed that the antagonistic potential of the two new species against this mushroom is perhaps equal to T. aggressivum. The P. ostreatus confrontation assays showed that T. pleuroticola has the highest affinity to overgrow mushroom mycelium among the green mold species. We conclude that the evolutionary pathway of T. pleuroticola could be in parallel to other saprotrophic and mycoparasitic species from the Harzianum clade and that this species poses the highest infection risk for mushroom farms, whereas T. pleurotum could be specialized for an ecological niche connected to components of Pleurotus substrata in cultivation. A DNA BarCode for identification of these species based on ITS1 and ITS2 sequences has been provided and integrated in the main database for Hypocrea/Trichoderma (www.ISTH.info).

BackgroundThe mitosporic fungus Trichoderma harzianum (Hypocrea, Ascomycota, Hypocreales, Hypocreaceae) is an ubiquitous species in the environment with some strains commercially exploited for the biological control of plant pathogenic fungi. Although T. harzianum is asexual (or anamorphic), its sexual stage (or teleomorph) has been described as Hypocrea lixii. Since recombination would be an important issue for the efficacy of an agent of the biological control in the field, we investigated the phylogenetic structure of the species.ResultsUsing DNA sequence data from three unlinked loci for each of 93 strains collected worldwide, we detected a complex speciation process revealing overlapping reproductively isolated biological species, recent agamospecies and numerous relict lineages with unresolved phylogenetic positions. Genealogical concordance and recombination analyses confirm the existence of two genetically isolated agamospecies including T. harzianum sensu stricto and two hypothetical holomorphic species related to but different from H. lixii. The exact phylogenetic position of the majority of strains was not resolved and therefore attributed to a diverse network of recombining strains conventionally called 'pseudoharzianum matrix'. Since H. lixii and T. harzianum are evidently genetically isolated, the anamorph - teleomorph combination comprising H. lixii/T. harzianum in one holomorph must be rejected in favor of two separate species.ConclusionsOur data illustrate a complex speciation within H. lixii - T. harzianum species group, which is based on coexistence and interaction of organisms with different evolutionary histories and on the absence of strict genetic borders between them.

Fungi that inhabit marine sponges occupy an ecological niche that has recently attracted great attention due to the potential in either ecological or pharmaceutical advances. The ecological interaction between marine sponges and fungi is, however, only poorly understood. Eighty five fungal taxa were isolated from the marine sponge Psammocinia sp. from the Mediterranean Sea. The majority (89%) of these taxa were isolated using a`sample compressing`method, in combination with the use of fungicides-amended medium. Abundant`terrestrial`taxa such as Acremonium, Penicillium and Trichoderma were found along with potentially undescribed Phoma and Trichoderma species. Several of these taxa exhibited in vitro anti-fungal properties as determined against four test fungi. Even though a significant number of fungal taxa were isolated during this study, we estimate that the diversity of fungi that are associated with Psammocinia sp. is higher than reported here. It is advocated that Psammocinia, and other sponge genera, may be a prime niche for discovering new fungal species as well as novel anti-fungal compounds from fungal sources.

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. INTRODUCTIONMany mould species are capable of causing invasive mycoses of mammals, including humans, under appropriate conditions, but of the more than one million fungal species which are estimated to exist in nature, only a few hundred have been associated with human and animal diseases (Walsh & Groll, 1999). In the last decade, however, case reports on infections by common mould fungi have increased, due to HIV/AIDS and the use of immunosuppressants for organ transplantation and cancer therapies. Species from the fungal genus Hypocrea/Trichoderma (Hypocreales, Ascomycota) have recently also joined this emerging list of such opportunistic pathogens. Detailed case reports of Trichoderma infections have been summarized by Kredics et al. (2003) Myoken et al. 2002). While Trichoderma isolates are still not a major threat, they nevertheless pose difficult diagnostic and therapeutic challeng...

Highlights► Longibrachiatum clade consists of at least 26 phylogenetic species. ► Many species are allopatric although sympatric species are also present. ► The majority of species lost their ability to sexual reproduction. ► The K/θ method is a useful measure to delineate species in the Longibrachiatum clade. ► The combination of the GCPSR and K/θ method gives the most adequate result for species delineation.

We have used a Mediterranean hot spot of biodiversity (the Island of Sardinia) to investigate the impact of abiotic factors on the distribution of species of the common soil fungus Trichoderma. To this end, we isolated 482 strains of Hypocrea/Trichoderma from 15 soils comprising undisturbed and disturbed environments (forest, shrub lands and undisturbed or extensively grazed grass steppes respectively). Isolates were identified at the species level by the oligonucleotide BarCode for Hypocrea/Trichoderma (TrichOKEY), sequence similarity analysis (Trichoblast) and phylogenetic inferences. The majority of the isolates were positively identified as pan-European and/or pan-global Hypocrea/Trichoderma species from sections Trichoderma and Pachybasium, comprising H. lixii/T. harzianum, T. gamsii, T. spirale, T. velutinum, T. hamatum, H. koningii/T. koningii, H. virens/T. virens, T. tomentosum, H. semiorbis, H. viridescens/T. viridescens, H. atroviridis/T. atroviride, T. asperellum, H. koningiopsis/T. koningiopsis and Trichoderma sp. Vd2. Only one isolate represented a new, undescribed species belonging to the Harzianum-Catoptron Clade. Internal transcribed spacer sequence analysis revealed only one potentially endemic internal transcribed spacer 1 allele of T. hamatum. All other species exhibited genotypes that were already found in Eurasia or in other continents. Only few cases of correlation of species occurrence with abiotic factors were recorded. The data suggest a strong reduction of native Hypocrea/Trichoderma diversity, which was replaced by extensive invasion of species from Eurasia, Africa and the Pacific Basin.

Abstract:Hypocrea/Trichoderma is a genus of soil-borne or wood-decaying fungi containing members important to mankind as producers of industrial enzymes and biocontrol agents against plant pathogens, but also as opportunistic pathogens of immunocompromised humans and animals, while others can cause damage to cultivated mushroom. With the recent advent of a reliable, BarCode-aided identification system for all known taxa of Trichoderma and Hypocrea, it became now possible to study some of the biological fundamentals of the diversity in this fungal genus in more detail. In this article, we will therefore review recent progress in (1) the understanding of the geographic distribution of individual taxa; (2) mechanisms of speciation leading to development of mushroom diseases and facultative human mycoses; and (3) the possible correlation of specific traits of secondary metabolism and molecular phylogeny.

The scarcity of fresh water in the Mediterranean region necessitates the search for halotolerant agents of biological control of plant diseases that can be applied in arid-zone agriculture irrigated with saline water. Among 29 Trichoderma strains previously isolated from Mediterranean Psammocinia sp. sponges, the greatest number of isolates belong to the Trichoderma longibrachiatum-Hypocrea orientalis species pair (9), H. atroviridis/T. atroviride (9), and T. harzianum species complex (7), all of which are known for high mycoparasitic potential. In addition, one isolate of T. asperelloides and two putative new species, Trichoderma sp. O.Y. 14707 and O.Y. 2407, from Longibrachiatum and Strictipilosa clades, respectively, have been identified. In vitro salinity assays showed that the ability to tolerate increasing osmotic pressure (halotolerance) is a strain-or clade-specific property rather than a feature of a species. Only a few isolates were found to be sensitive to increased salinity, while others either were halotolerant or even demonstrated improved growth in increasingly saline conditions. In vitro antibiosis assays revealed strong antagonistic activity toward phytopathogens due to the production of both soluble and volatile metabolites. Two marine-derived Trichoderma isolates, identified as T. atroviride and T. asperelloides, respectively, effectively reduced Rhizoctonia solani damping-off disease on beans and also induced defense responses in cucumber seedlings against Pseudomonas syringae pv. lachrimans. This is the first inclusive evaluation of marine fungi as potential biocontrol agents.