Harzianum Clade Research Articles

The neutral metallopeptidase NMP1 of Trichoderma guizhouense is required for mycotrophy and self-defence.

Trichoderma guizhouense NJAU 4742 (Harzianum clade) can suppress the causative agent of banana wild disease Fusarium oxysporum f. sp. cubense 4 (Foc4). To identify genes involved in this trait, we used T-DNA insertional mutagenesis and isolated one mutant that was unable to overgrow Foc4 and had reduced antifungal ability. Using the high-efficiency thermal asymmetric interlaced-PCR, the T-DNA was located in the terminator of a neutral metalloprotease gene (encoding a MEROPS family M35 protease), which was named nmp1. The antifungal activity of the mutant was recovered by retransformation with wild-type nmp1 gene. The purified NMP1 (overexpressed in Pichia pastoris) did not inhibit the growth and germination of other fungi in vitro. Its addition, however, partly recovered the antifungal activity of the mutant strain against some fungi. The expression of nmp1 is induced by the presence of fungi and by dead fungal biomass, but the time-course of transcript accumulation following the physical contact depends on mode of interaction: it increases in cases of long-lasting parasitism and decreases if the prey fungus is dead shortly after or even before the contact (predation). We thus conclude that NMP1 protein of T. guizhouense has major importance for mycotrophic interactions and defence against other fungi.

Trichoderma (Hypocrea) species with green ascospores from China.

Stromata of Trichoderma species having green ascospores were collected in various regions of China. Based on morphology of the sexual and asexual morph, culture characteristics, and sequence analyses of rpb2 and tef1 genes, 17 species with green ascospores were identified. Among them, Trichoderma rosulatum, T. rufobrunneum and T. stipitatum are described as new species, and seven other species are reported for the first time from China. Trichoderma rosulatum produces small bright yellow or pale greenish stromata with dense dark green ostioles and gliocladium-like conidiophores, shows a close relationship to T. thelephoricola, and belongs to the Chlorospora clade. Trichoderma rufobrunneum, which typically forms reddish brown stromata, is recognised as a member of the Harzianum clade. Trichoderma stipitatum is characterised by turbinate, pale yellow to nearly orange stromata and verticillium-like conidiophores; it forms a distinct, independent lineage with strong bootstrap support in the phylogenetic trees. The distinctions between the new species and their close relatives are discussed, and their phylogenetic positions are explored.

Ribosomal DNA Sequence Based Characterization of Trichoderma Isolates Antagonistic to Colletotrichum falcatum Causing Red Rot Disease of Sugarcane

Trichoderma bioagent isolates cultured from the sugarcane rhizosphere of subtropical India, and established efficient against Colletotrichum falcatum Went (red rot pathogen) in sugarcane are described based on phylogenetic analysis of nucleotide sequences. DNA sequences of the isolates included the internal transcribed spacer (ITS) regions of the rDNA cluster (ITS1-5.8S- ITS2). The 5.8S region was found conserved and much of the sequence variability was due to indels or transition/transversion mutations in the ITS1 and 2 regions. Both the isolates belonged to the T. harzianum clade. These isolates are distinct yet have close genetic similarity with several established strains/isolates of T. harzianum reported worldwide. Correct identification of these bioagents would augment the effective utilization of these fungi of immense agricultural importance in managing the red rot disease of sugarcane.

Trichoderma amazonicum, a new endophytic species on Hevea brasiliensis and H. guianensis from the Amazon basin

A new species of Trichoderma (teleomorph Hypocrea, Ascomycota, Sordariomycetes, Hypocreales, Hypocreaceae), T. amazonicum, endophytic on the living sapwood and leaves of Hevea spp. trees is described. Trichoderma amazonicum is distinguished from closely related species in the Harzianum clade (e.g. Hypocrea alni, H. brunneoviridis, H. epimyces, H. parepimyces, T. aggressivum, T. harzianum, T. pleuroticola and T. pleuroti) by morphological and ecological characteristics and phylogenetic analysis of three loci (ITS nrDNA, tef1 and rpb2). The closest relatives of this species are the facultatively fungicolous species T. pleuroticola and T. pleuroti.

MALDI-TOF MS of Trichoderma: a model system for the identification of microfungi

This investigation aimed to assess whether MALDI-TOF MS analysis of the proteome could be applied to the study of Trichoderma, a fungal genus selected because it includes many species and is phylogenetically well defined. We also investigated whether MALDI-TOF MS analysis of peptide mass fingerprints would reveal apomorphies that could be useful in diagnosing species in this genus. One hundred and twenty nine morphologically and genetically well-characterized strains of Hypocrea and Trichoderma, belonging to 25 species in 8 phylogenetic clades, were analyzed by MALDI-TOF MS mass spectrometry. The resulting peak lists of individual samples were submitted to single-linkage cluster analysis to produce a taxonomic tree and were compared to ITS and tef1 sequences from GenBank. SuperSpectra™ for the 13 most relevant species of Trichoderma were computed. The results confirmed roughly previously defined clades and sections. With the exceptions of T. saturnisporum (Longibrachiatum Clade) and T. harzianum (Harzianum Clade), strains of individual species clustered very closely. T. polysporum clustered distantly from all other groups. The MALDI-TOF MS analysis accurately reflected the phylogenetic classification reported in recent publications, and, in most cases, strains identified by DNA sequence analysis clustered together by MALDI-TOF MS. The resolution of MALDI-TOF MS, as performed here, was roughly equivalent to ITS rDNA. The MALDI-TOF MS technique analyzes peptides and represents a rough equivalent to sequencing, making this method a useful adjunct for determination of species limits. It also allows simple, reliable, and quick species identification, thus representing a valid alternative to gene sequencing for species diagnosis of Trichoderma and other fungal taxa.

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.

Genetically closely related but phenotypically divergent Trichoderma species cause green mold disease in oyster mushroom farms worldwide.

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).

Two new species of Trichoderma from Yunnan, China

Two new species of the fungal genus Trichoderma, Trichoderma compactum and Trichoderma yunnanense, isolated from rhizosphere of tobacco in Yunnan Province, China are described based on morphological characters and phylogenetic analyses of nucleotide sequences. Our DNA sequences included the internal transcribed spacer (ITS) regions of the rDNA cluster (ITS1 and ITS2), and partial sequences of the translation elongation factor 1-alpha (tef1) and a fragment of the gene coding for endochitinase 42 (ech42). The analyses show that T. compactum belongs to the Harzianum clade, and T. yunnanense belongs to the Hamatum clade.

Identification ofTrichoderma biocontrol isolates to clades according to ap-PCR and ITS sequence analyses

A collection ofTrichoderma isolates, with different biocontrol capabilities, were identified by molecular methods. Arbitrarily-primed PCR (ap-PCR) using repeat motif primers was performed on DNA from aTrichoderma spp. collection of 76 isolates, and representative isolates were further characterized into three main clades by internal transcribed spacer (ITS) sequence analysis. Consequently, a reliable phylogenetic tree was constructed containing isolates belonging to theT. harzianum clade (comprisingT. aureoviride, T. inhamatum, andT. virens), theT. longibrachiatum andT. saturnisporum cluster, and that including the speciesT. asperellum, T. atroviride, T. koningii andT. viride.