SCAR-based real time PCR to identify a biocontrol strain (T1) of Trichoderma atroviride and study its population dynamics in soils

Strains of Trichoderma spp. are known for their antagonistic properties against plant pathogens, some are already on the market, others are under development. In order to launch a strain on the market its perfect identification at the species and strain levels is needed. The aim of this study is to (i) design a SCAR marker for specific identification of strain T1 of Trichoderma atroviride and (ii) monitor population dynamics of this strain in soil by real time PCR. A primer pair targeting a 141-bp fragment enabled specific detection of this strain without cross detection of autochthonous populations of Trichoderma in several field soils. In two soils, population dynamics assessed by real time PCR and the soil plate technique gave similar results. The molecular tools developed in this study satisfy the requirement for specific identification of the biocontrol strain and for detection and quantification of T. atroviride T1 population in complex environments.     

Evaluating the survival and environmental fate of the biocontrol agent Trichoderma atroviride SC1 in vineyards in northern Italy

Aims:  To study the survival in the soil and the dispersion in the environment of Trichoderma atroviride SC1 after soil applications in a vineyard.
Methods and Results:  Trichoderma atroviride SC1 was introduced into soil in two consecutive years. The levels of T. atroviride populations at different spatial and temporal points following inoculation were assessed by counting the colony-forming units and by a specific quantitative real-time PCR. A high concentration of T. atroviride SC1 was still observed at the 18th week after inoculation. The vertical migration of the fungus to a soil depth of 0·4 m was already noticeable during the first week after inoculation. The fungus spread up to 4 m (horizontally) from the point of inoculation and its concentration decreased with the increasing distance (horizontal and vertical). It was able to colonize the rhizosphere and was also found on grapevine leaves. One year after soil inoculation, T. atroviride SC1 could still be recovered in the treated areas.
Conclusions:  Trichoderma atroviride SC1 survived and dispersed becoming an integrant part of the local microbial community under the tested conditions.
Significance and Impact of the Study:  The persistence and rapid spread of T. atroviride SC1 represent good qualities for its future use as biocontrol agent against soilborne pathogens.     

An intact soil-core microcosm method to evaluate the survival and vertical dispersal of Trichoderma atroviride SC1

Aim:  To develop an intact soil-core microcosm method to study the survival and vertical dispersal of an experimental biocontrol agent ( Trichoderma atroviride SC1) applied to the soil surface.
Methods and Results:  The soil for the microcosms was collected using iron pipes with perforations corresponding to different soil layers. The tool was inserted into the soil and gently removed with the soil core inside. Trichoderma atroviride SC1 was mixed with the top layer of soil in the pipe. The experiment was performed in 2006 and 2007, and data from the microcosms were compared with results obtained under field conditions in the locations in which, the microcosms were collected, in the same periods. The concentrations of T. atroviride SC1 in the soil were estimated immediately after treatment, and 1, 5, 9 and 18 weeks after treatment at both the soil surface and the above-mentioned depths. The development of T. atroviride SC1 populations in the microcosms during the 18 weeks of monitoring was similar to that observed under field conditions. The dispersal of conidia was affected by the application of water to the soil.
Conclusions:  Results demonstrate that this microcosm prototype can be used to model the behaviour of T. atroviride SC1 in soil.
Significance and Impact of the Study:  The intact soil-core microcosm is a reliable, easy-to-use, fast and cheap method that could also be used in studies of similar filamentous fungi to study their probable fate in the soil prior to their being introduced into the environment.     

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.     

A new cultivation independent approach to detect and monitor common Trichoderma species in soils

A set of primers was developed for the detection, identification and quantification of common Trichoderma species in soil samples. Based on a broad range master alignment primers were derived to amplify an approximate 540 bp fragment comprising the internal transcribed spacer region 1 (ITS 1), 5.8S rDNA and internal transcribed spacer region 2 (ITS 2) from all taxonomic Clades of the genus Trichoderma. The primer set was applied to test strains as well as community DNA isolated from arable and forest soil. For all tested isolates the corresponding internal transcribed spacer regions of Trichoderma spp. strains were amplified, but none of non-Trichoderma origin. PCR with community DNA from soil yielded products of the expected size. Analysis of a clone library established for an arable site showed that all amplified sequences originated exclusively from Trichoderma species mainly being representatives of the Clades Hamatum, Harzianum and Pachybasioides and comprising most of the species known for biocontrol ability. In a realtime PCR approach the primer set uTf/uTr also proved to be a suitable system to quantify DNA of Trichoderma spp. in soils.     

洞庭湖湿地木霉多样性及生防活性

【目的】了解湖南省洞庭湖湿地木霉种类及分布,丰富我国的木霉种质资源,为功能菌株筛选应用奠定基础。【方法】利用ITS序列比对分析结合形态学特征对分离到的木霉菌株进行种类鉴定,构建系统发育进化树分析其亲缘关系。通过菌丝生长速率法测定菌株的平板抑菌能力,根据水解带大小检测菌株的水解酶活性,利用灰色关联度分析筛选综合生防效果较好的木霉菌株。【结果】从52个土样和18个水样中分离得到114株木霉菌株,经鉴定分属16个木霉种类:哈茨木霉、绿木霉、刺孢木霉、土星孢木霉、钩状木霉、拟康宁木霉、短密木霉、深绿木霉、猥木霉、毛细木霉(中国新记录种)、长枝木霉、卵孢木霉、侧耳木霉、加纳木霉、厚木霉及一个疑似新种;哈茨木霉为洞庭湖湿地中的优势种,占总菌株数量的19.30%;16种木霉在系统发育树中归于7个进化支:Harzianum Clade、Virens Clade、Longibrachiatum Clade、Lutea Clade、Viride Clade、Hamatum Clade、Unknown Clade。灰色关联度分析表明,菌株TW21990、QT22040和QT22094的灰色关联度较高,分别为0.849 5、0.798 6和0.732 6,综合生防性状较好。【结论】洞庭湖湿地木霉具有种类多样性和分布多样性,发现了一个中国新记录种毛细木霉和一个疑似新种,哈茨木霉TW21990、长枝木霉QT22040和卵孢木霉QT22094是潜在的优良生防菌株。     

A microsatellite based method for quantification of fungi in decomposing plant material elucidates the role of Fusarium graminearum DON production in the saprophytic competition with Trichoderma atroviride in maize tissue microcosms

Common PCR assays for quantification of fungi in living plants cannot be used to study saprophytic colonization of fungi because plant decomposition releases PCR-inhibiting substances and saprophytes degrade the plant DNA which could serve as internal standard. The microsatellite PCR assays presented here overcome these problems by spiking samples prior to DNA extraction with mycelium of a reference strain. PCR with fluorescent primers co-amplifies microsatellite fragments of different length from target and reference strains. These fragments were separated in a capillary sequencer with fluorescence detection. The target/reference ratio of fluorescence signal was used to calculate target biomass in the sample. Such PCR assays were developed for the mycotoxin deoxynivalenol (DON)-producing wheat and maize pathogen Fusarium graminearum and the biocontrol agent Trichoderma atroviride, using new microsatellite markers. In contrast to real-time PCR assays, the novel PCR assays showed reliable fungal biomass quantification in samples with differentially decomposed plant tissue. The PCR assays were used to quantify the two fungi after competitive colonization of autoclaved maize leaf tissue in microcosms. Using a DON-producing F. graminearum wild-type strain and its nontoxigenic mutant we found no evidence for a role of DON production in F. graminearum defense against T. atroviride. The presence of T. atroviride resulted in a 36% lower wild-type DON production per biomass.     

Detection of the biocontrol agent Colletotrichum coccodes (183088) from the target weed velvetleaf and from soil by strain-specific PCR markers

Diagnostic molecular markers, generated from random amplified polymorphic DNA (RAPD) and used in polymerase chain reaction (PCR), were developed to selectively recognize and detect the presence of a single strain of the biocontrol fungus Colletotrichum coccodes (183088) on the target weed species Abutilon theophrasti and from soil samples. Several isolates of C. coccodes, 15 species of Colletotrichum, a variety of heterogeneous organisms and various plant species were first screened by RAPD-PCR, and a strain specific marker was identified for C. coccodes (183088). No significant sequence similarity was found between this marker and any other sequences in the databases. The marker was converted into a sequence-characterised amplified region (SCAR), and specific primer sets (N5F/N5R, N5Fi/N5Ri) were designed for use in PCR detection assays. The primer sets N5F/N5R and N5Fi/N5Ri each amplified a single product of 617 and 380 bp, respectively, with DNA isolated from strain 183088. The specificity of the primers was confirmed by the absence of amplified products with DNA from other C. coccodes isolates, other species representing 15 phylogenetic groups of the genus Colletotrichum and 11 other organisms. The SCAR primers (N5F/N5R) were successfully used to detect strain 183088 from infected velvetleaf plants but not from seeded greenhouse soil substrate or from soil samples originating from deliberate-released field experiments. The sensitivity of the assay was substantially increased 1000-fold when nested primers (N5Fi/N5Ri) were used in a second PCR run. N5Fi/N5Ri selectively detected strain 183088 from seeded greenhouse soils as well as from deliberate-released field soil samples without any cross-amplification with other soil microorganisms. This rapid PCR assay allows an accurate detection of C. coccodes strain 183088 among a background of soil microorganisms and will be useful for monitoring the biocontrol when released into natural field soils.     

Pseudomonas lipodepsipeptides and fungal cell wall-degrading enzymes act synergistically in biological control

Pseudomonas syringae pv. syringae strain B359 secreted two main lipodepsipeptides (LDPs), syringomycin E (SRE) and syringopeptin 25A (SP25A), together with at least four types of cell wall-degrading enzymes (CWDEs). In antifungal bioassays, the purified toxins SRE and SP25A interacted synergistically with chitinolytic and glucanolytic enzymes purified from the same bacterial strain or from the biocontrol fungus Trichoderma atroviride strain P1. The synergism between LDPs and CWDEs occurred against all seven different fungal species tested and P. syringae itself, with a level dependent on the enzyme used to permeabilize the microbial cell wall. The antifungal activity of SP25A was much more increased by the CWDE action than was that of the smaller SRE, suggesting a stronger antifungal role for SP25A. In vivo biocontrol assays were performed by using P. syringae alone or in combination with T. atroviride, including a Trichoderma endochitinase knock-out mutant in place of the wild type and a chitinase-specific enzyme inhibitor. These experiments clearly indicate that the synergistic interaction LDPs-CWDEs is involved in the antagonistic mechanism of P. syringae, and they support the concept that a more effective disease control is given by the combined action of the two agents.     

Improvement of the fungal biocontrol agent Trichoderma atroviride to enhance both antagonism and induction of plant systemic disease resistance

Biocontrol agents generally do not perform well enough under field conditions to compete with chemical fungicides. We determined whether transgenic strain SJ3-4 of Trichoderma atroviride, which expresses the Aspergillus niger glucose oxidase-encoding gene, goxA, under a homologous chitinase (nag1) promoter had increased capabilities as a fungal biocontrol agent. The transgenic strain differed only slightly from the wild-type in sporulation or the growth rate. goxA expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted. SJ3-4 had significantly less N-acetylglucosaminidase and endochitinase activities than its nontransformed parent. Glucose oxidase-containing culture filtrates exhibited threefold-greater inhibition of germination of spores of Botrytis cinerea. The transgenic strain also more quickly overgrew and lysed the plant pathogens Rhizoctonia solani and Pythium ultimum. In planta, SJ3-4 had no detectable improved effect against low inoculum levels of these pathogens. Beans planted in heavily infested soil and treated with conidia of the transgenic Trichoderma strain germinated, but beans treated with wild-type spores did not germinate. SJ3-4 also was more effective in inducing systemic resistance in plants. Beans with SJ3-4 root protection were highly resistant to leaf lesions caused by the foliar pathogen B. cinerea. This work demonstrates that heterologous genes driven by pathogen-inducible promoters can increase the biocontrol and systemic resistance-inducing properties of fungal biocontrol agents, such as Trichoderma spp., and that these microbes can be used as vectors to provide plants with useful molecules (e.g., glucose oxidase) that can increase their resistance to pathogens.     

In vivo study of trichoderma-pathogen-plant interactions, using constitutive and inducible green fluorescent protein reporter systems

Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.     

An oligonucleotide barcode for species identification in Trichoderma and Hypocrea

One of the biggest obstructions to studies on Trichoderma has been the incorrect and confused application of species names to isolates used in industry, biocontrol of plant pathogens and ecological surveys, thereby making the comparison of results questionable. Here we provide a convenient, on-line method for the quick molecular identification of Hypocrea/Trichoderma at the genus and species levels based on an oligonucleotide barcode: a diagnostic combination of several oligonucleotides (hallmarks) specifically allocated within the internal transcribed spacer 1 and 2 (ITS1 and 2) sequences of the rDNA repeat. The barcode was developed on the basis of 979 sequences of 88 vouchered species which displayed in total 135 ITS1 and 2 haplotypes. Oligonucleotide sequences which are constant in all known ITS1 and 2 of Hypocrea/Trichoderma but different in closely related fungal genera, were used to define genus-specific hallmarks. The library of species-, clade- and genus-specific hallmarks is stored in the MySQL database and integrated in the TrichOKey v. 1.0 - barcode sequence identification program with the web interface located on . TrichOKey v. 1.0 identifies 75 single species, 5 species pairs and 1 species triplet. Verification of the DNA-barcode was done by a blind test on 53 unknown isolates of Trichoderma, collected in Central and South America. The obtained results were in a total agreement with phylogenetic identification based on tef1 (large intron), NCBI BLAST of vouchered records and postum morphological analysis. We conclude that oligonucleotide barcode is a powerful tool for the routine identification of Hypocrea/Trichoderma species and should be useful as a complement to traditional methods.     

Specific detection of Lysobacter enzymogenes (Christensen and Cook 1978) strain 3.1T8 with TaqMan® PCR

Aims: To develop a strain‐specific TaqMan^® PCR method for detecting and quantifying the biocontrol strain Lysobacter enzymogenes 3.1T8. Methods and Results: A primer–probe combination was designed on the basis of a strain‐specific sequence selected using REP‐PCR (repetitive extragenic palindromic‐polymerase chain reaction). The specificity of this combination was demonstrated by 14 other Lysobacter strains that did not react with the selected primer–probe combination. To quantify strain 3.1T8 in cucumber root samples, a calibration curve was prepared by spiking roots with a 10‐fold dilution series of the strain. Detection of the biocontrol strain 3.1T8 with this method showed that the strain survived well for 22 days on root tips as well as on older cucumber roots. Survival was higher when the strain was inoculated to younger plants. In a cucumber production system with large volumes of substrate, strain 3.1T8 was detected in high numbers on cucumber roots 3 weeks after inoculation. Conclusions: The primer–probe combination developed was strain specific, because it did not react with other strains of the same species and genus. The TaqMan^® PCR method successfully quantified the inoculated biocontrol strain on cucumber roots grown in different cropping systems. Significance and Impact of the Study: The developed TaqMan^® PCR method is a strain‐specific real‐time detection method that can be used to assess the population dynamics of L. enzymogenes strain 3.1T8 for further optimization of its biocontrol efficacy.     

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

Background

Mycoparasitism, 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.

Results

Here 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.

Conclusions

The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.     

Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates

We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting species-specific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.     

Molecular identification of Trichoderma species associated with Pleurotus ostreatus and natural substrates of the oyster mushroom

Green mold of Pleurotus ostreatus , caused by Trichoderma species, has recently resulted in crop losses worldwide. Therefore, there is an emerging need for rapid means of diagnosing the causal agents. A PCR assay was developed for rapid detection of Trichoderma pleurotum and Trichoderma pleuroticola , the two pathogens causing green mold of P. ostreatus . Three oligonucleotide primers were designed for identifying these species in a multiplex PCR assay based on DNA sequences within the fourth and fifth introns in the translation elongation factor 1α gene. The primers detected the presence of T. pleurotum and/or T. pleuroticola directly in the growing substrates of oyster mushrooms, without the need for isolating the pathogens. The assay was used to assess the presence of the two species in natural environments in which P. ostreatus can be found in Hungary, and demonstrated that T. pleuroticola was present in the growing substrates and on the surface of the basidiomes of wild oyster mushrooms. Other Trichoderma species detected in these substrates and habitats were Trichoderma harzianum, Trichoderma longibrachiatum and Trichoderma atroviride. Trichoderma pleurotum was not found in any of the samples from the forested areas tested in this study.     

Effect of Trichoderma species fungi on soil micromycetes, causing infectious conifer seedling lodging in Siberian tree nurseries

Soils in the tree nurseries studied were characterized by a lower species diversity of fungi than adjacent virgin soils. In particular, the relative abundances of representatives of the genera Mucor, Chaetomium, and Trichoderma in the nursery soil were two times lower than in adjacent virgin soils. On the other hand, the nursery soil exhibited greater abundances of fungi of the genus Fusarium, which are causative agents of many diseases of conifer seedlings. To appreciate the efficiency of biocontrol of the infectious diseases of conifer seedlings, we introduced several indigenous Trichoderma strains into the nursery soil and found that this affected the species composition of soil microflora considerably. Changes in the species composition of mycobiota beneficially influenced the phytosanitary state of soils and reduced the infectious lodging of conifer seedlings.     

Identification of Trichoderma strains by image analysis of HPLC chromatograms

Forty-four Trichoderma strains from water-damaged building materials or indoor dust were classified with chromatographic image analysis on full chromatographic matrices obtained by high performance liquid chromatography with UV detection of culture extracts. The classes were compared with morphological identification and rDNA sequence data, and for each class all strains were of the same identity. With all three techniques each strain--except one--was identified as the same species. These strains belonged to Trichoderma atroviride (nine strains), Trichoderma viride (three strains), Trichoderma harzianum (10 strains), Trichoderma citrinoviride (12 strains), and Trichoderma longibrachiatum (nine strains). The odd strain was identified as Trichoderma hamatum by morphology and rDNA sequencing, but not by image analysis as no reference strains of this species were included. It is concluded that the secondary metabolite profile contains sufficient information for classification and species identification.     

Signal transduction by Tga3, a novel G protein alpha subunit of Trichoderma atroviride

Trichoderma species are used commercially as biocontrol agents against a number of phytopathogenic fungi due to their mycoparasitic characterisitics. The mycoparasitic response is induced when Trichoderma specifically recognizes the presence of the host fungus and transduces the host-derived signals to their respective regulatory targets. We made deletion mutants of the tga3 gene of Trichoderma atroviride, which encodes a novel G protein alpha subunit that belongs to subgroup III of fungal Galpha proteins. Deltatga3 mutants had changes in vegetative growth, conidiation, and conidial germination and reduced intracellular cyclic AMP levels. These mutants were avirulent in direct confrontation assays with Rhizoctonia solani or Botrytis cinerea, and mycoparasitism-related infection structures were not formed. When induced with colloidal chitin or N-acetylglucosamine in liquid culture, the mutants had reduced extracellular chitinase activity even though the chitinase-encoding genes ech42 and nag1 were transcribed at a significantly higher rate than they were in the wild type. Addition of exogenous cyclic AMP did not suppress the altered phenotype or restore mycoparasitic overgrowth, although it did restore the ability to produce the infection structures. Thus, T. atroviride Tga3 has a general role in vegetative growth and can alter mycoparasitism-related characteristics, such as infection structure formation and chitinase gene expression.     

Recent advances and future prospects in peptaibiotics, hydrophobin, and mycotoxin research, and their importance for chemotaxonomy of Trichoderma and Hypocrea

Fungi of the genus Trichoderma with teleomorphs in Hypocrea are abundant producers of a group of amphiphilic, non-ribosomal peptide antibiotics, which are rich in the non-proteinogenic amino acid Aib (alpha-aminoisobutyric acid). They are referred to as peptaibiotics, or peptaibols, if a 1,2-amino alcohol is present at the C-terminus. Trichoderma/Hypocrea, like other ascomycetous fungi, also produce hydrophobins, a class of small, cysteine-rich proteins. Advanced soft ionization mass spectrometric techniques such as LC-CID-MS, LC-ESI-MS(n), and IC-MALDI-TOF-MS enabled the high-throughput analysis, simultaneous detection and sequence determination of peptaibiotics and hydrophobins from minute quantities of fungal materials. Some Trichoderma species have been recognized to produce peptaibiotics as well as simple mycotoxins of the trichothecene group. The combination of sequence data of both groups of peptides with the pattern of low-molecular-weight secondary metabolites, including trichothecene-type mycotoxins, independently confirmed the results of morphological, molecular, and phylogenetic analyses. This approach established a new lineage in Trichoderma/Hypocrea, the Brevicompactum clade, comprising four new and one redescribed species. Notably, commercial preparations of single or mixed cultures of Trichoderma species, in particular T. harzianum, and T. koningii, are registered as biocontrol agents for soil and plant pathogens. In this context, it is emphasized that the four mycotoxin-producing species of the recently established Brevicompactum clade (T. brevicompactum, T. arundinaceum, T. turrialbense, and T. protrudens) are not closely related to any of the Trichoderma species currently used as biocontrol agents. Furthermore, possible health concerns about release of peptaibiotics in the biosphere are discussed with respect to their bioactivities and their use as drugs in human and veterinary medicine. Finally, future prospects regarding novel bioactivities and further research needs, including interdisciplinary taxonomic approaches, are outlined.