DNA barcoding survey of Trichoderma diversity in soil and litter of the Colombian lowland Amazonian rainforest reveals Trichoderma strigosellum sp. nov. and other species.

The diversity of Trichoderma (Hypocreales, Ascomycota) colonizing leaf litter as well as the rhizosphere of Garcinia macrophylla (Clusiaceae) was investigated in primary and secondary rain forests in Colombian Amazonia. DNA barcoding of 107 strains based on the internal transcribed spacers 1 and 2 (ITS1 and 2) of the ribosomal RNA gene cluster and the partial sequence of the translation elongation factor 1 alpha (tef1) gene revealed that the diversity of Trichoderma was dominated (71 %) by three common cosmopolitan species, namely Trichoderma harzianum sensu lato (41 %), Trichoderma spirale (17 %) and Trichoderma koningiopsis (13 %). Four ITS 1 and 2 phylotypes (13 strains) could not be identified with certainty. Multigene phylogenetic analysis and phenotype profiling of four strains with an ITS1 and 2 phylotype similar to Trichoderma strigosum revealed a new sister species of the latter that is described here as Trichoderma strigosellum sp. nov. Sequence similarity searches revealed that this species also occurs in soils of Malaysia and Cameroon, suggesting a pantropical distribution.

Molecular phylogeny and species delimitation in the section Longibrachiatum of Trichoderma.

The phylogenetically most derived group of the genus Trichoderma - section Longibrachiatum, includes some of the most intensively studied species, such as the industrial cellulase producer T. reesei (teleomorph Hypocrea jecorina), or the facultative opportunistic human pathogens T. longibrachiatum and H. orientalis. At the same time, the phylogeny of this clade is only poorly understood. Here we used a collection of 112 strains representing all currently recognized species and isolates that were tentatively identified as members of the group, to analyze species diversity and molecular evolution. Bayesian phylogenetic analyses based on several unlinked loci in individual and concatenated datasets confirmed 13 previously described species and 3 previously recognized phylogenetic species all of which were not yet described formally. When the genealogical concordance criterion, the K/θ method and comparison of frequencies of pairwise nucleotide differences were applied to the data sample, 10 additional new phylogenetic species were recognized, seven of which consisted only of a single lineage. Our analysis thus identifies 26 putative species in section Longibrachiatum, what doubles the currently estimated taxonomic diversity of the group, and illustrates the power of combining genealogical concordance and population genetic analysis for dissecting species in a recently diverged group of fungal species.

Marine isolates of Trichoderma spp. as potential halotolerant agents of biological control for arid-zone agriculture.

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.

Muscodor fengyangensis sp. nov. from southeast China: morphology, physiology and production of volatile compounds.

The fungal genus Muscodor was erected on the basis of Muscodor albus, an endophytic fungus originally isolated from Cinnamomum zeylanicum. It produces a mixture of volatile organic compounds (VOCs) with antimicrobial activity that can be used as mycofumigants. The genus currently comprises five species. Here we describe the isolation and characterization of a new species of Muscodor on the basis of five endophytic fungal strains from leaves of Actinidia chinensis, Pseudotaxus chienii and an unidentified broad leaf tree in the Fengyangshan Nature Reserve, Zhejiang Province, Southeast of China. They exhibit white colonies on potato dextrose agar (PDA) media, rope-like mycelial strands, but did not sporulate. The optimum growth temperature is 25°C. The results of a phylogenetic analysis based on four loci (ITS1-5.8S-ITS2, 28S rRNA, rpb2 and tub1) are consistent with the hypothesis that these five strains belong to a single taxon. All five strains also produce volatile chemical components with antimicrobial activity in vitro, which were different from those previously described for other Muscodor species.

Clonal species Trichoderma parareesei sp. nov. likely resembles the ancestor of the cellulase producer Hypocrea jecorina/T. reesei.

We have previously reported that the prominent industrial enzyme producer Trichoderma reesei (teleomorph Hypocrea jecorina; Hypocreales, Ascomycota, Dikarya) has a genetically isolated, sympatric sister species devoid of sexual reproduction and which is constituted by the majority of anamorphic strains previously attributed to H. jecorina/T. reesei. In this paper we present the formal taxonomic description of this new species, T. parareesei, complemented by multivariate phenotype profiling and molecular evolutionary examination. A phylogenetic analysis of relatively conserved loci, such as coding fragments of the RNA polymerase B subunit II (rpb2) and GH18 chitinase (chi18-5), showed that T. parareesei is genetically invariable and likely resembles the ancestor which gave raise to H. jecorina. This and the fact that at least one mating type gene of T. parareesei has previously been found to be essentially altered compared to the sequence of H. jecorina/T. reesei indicate that divergence probably occurred due to the impaired functionality of the mating system in the hypothetical ancestor of both species. In contrast, we show that the sexually reproducing and correspondingly more polymorphic H. jecorina/T. reesei is essentially evolutionarily derived. Phenotype microarray analyses performed at seven temperature regimens support our previous speculations that T. parareesei possesses a relatively high opportunistic potential, which probably ensured the survival of this species in ancient and sustainable environment such as tropical forests.

The Trichoderma harzianum demon: complex speciation history resulting in coexistence of hypothetical biological species, recent agamospecies and numerous relict lineages.

BACKGROUND: The 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. RESULTS: Using 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. CONCLUSIONS: Our 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.

Evolution and ecophysiology of the industrial producer Hypocrea jecorina (Anamorph Trichoderma reesei) and a new sympatric agamospecies related to it.

BACKGROUND: Trichoderma reesei, a mitosporic green mould, was recognized during the WW II based on a single isolate from the Solomon Islands and since then used in industry for production of cellulases. It is believed to be an anamorph (asexual stage) of the common pantropical ascomycete Hypocrea jecorina. METHODOLOGY/PRINCIPAL FINDINGS: We combined molecular evolutionary analysis and multiple methods of phenotype profiling in order to reveal the genetic relationship of T. reesei to H. jecorina. The resulting data show that the isolates which were previously identified as H. jecorina by means of morphophysiology and ITS1 and 2 (rRNA gene cluster) barcode in fact comprise several species: i) H. jecorina/T. reesei sensu stricto which contains most of the teleomorphs (sexual stages) found on dead wood and the wild-type strain of T. reesei QM 6a; ii) T. parareesei nom. prov., which contains all strains isolated as anamorphs from soil; iii) and two other hypothetical new species for which only one or two isolates are available. In silico tests for recombination and in vitro mating experiments revealed a history of sexual reproduction for H. jecorina and confirmed clonality for T. parareesei nom. prov. Isolates of both species were consistently found worldwide in pantropical climatic zone. Ecophysiological comparison of H. jecorina and T. parareesei nom. prov. revealed striking differences in carbon source utilization, conidiation intensity, photosensitivity and mycoparasitism, thus suggesting adaptation to different ecological niches with the high opportunistic potential for T. parareesei nom. prov. CONCLUSIONS: Our data prove that T. reesei belongs to a holomorph H. jecorina and displays a history of worldwide gene flow. We also show that its nearest genetic neighbour--T. parareesei nom. prov., is a cryptic phylogenetic agamospecies which inhabits the same biogeographic zone. These two species thus provide a so far rare example of sympatric speciation within saprotrophic fungi, with divergent ecophysiological adaptations and reproductive strategies.

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 1alpha 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.

Differential regulation and posttranslational processing of the class II hydrophobin genes from the biocontrol fungus Hypocrea atroviridis.

Hydrophobins are small extracellular proteins, unique to and ubiquitous in filamentous fungi, which mediate interactions between the fungus and environment. The mycoparasitic fungus Hypocrea atroviridis has recently been shown to possess 10 different class II hydrophobin genes, which is a much higher number than that of any other ascomycete investigated so far. In order to learn the potential advantage of this hydrophobin multiplicity for the fungus, we have investigated their expression patterns under different physiological conditions (e.g., vegetative growth), various conditions inducing sporulation (light, carbon starvation, and mechanical injury-induced stress), and confrontation with potential hosts for mycoparasitism. The results show that the 10 hydrophobins display different patterns of response to these conditions: one hydrophobin (encoded by hfb-2b) is constitutively induced under all conditions, whereas other hydrophobins were formed only under conditions of carbon starvation (encoded by hfb-1c and hfb-6c) or light plus carbon starvation (encoded by hfb-2c, hfb-6a, and hfb-6b). The hydrophobins encoded by hfb-1b and hfb-5a were primarily formed during vegetative growth and under mechanical injury-provoked stress. hfb-22a was not expressed under any conditions and is likely a pseudogene. None of the 10 genes showed a specific expression pattern during mycoparasitic interaction. Most, but not all, of the expression patterns under the three different conditions of sporulation were dependent on one or both of the two blue-light regulator proteins BLR1 and BLR2, as shown by the use of respective loss-of-function mutants. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of mycelial solvent extracts provided sets of molecular ions corresponding to HFB-1b, HFB-2a, HFB-2b, and HFB-5a in their oxidized and processed forms. These in silico-deduced sequences of the hydrophobins indicate cleavages at known signal peptide sites as well as additional N- and C-terminal processing. Mass peaks observed during confrontation with plant-pathogenic fungi indicate further proteolytic attack on the hydrophobins. Our study illustrates both divergent and redundant functions of the 10 hydrophobins of H. atroviridis.

Fungal diversity in the rhizosphere of endemic plant species of Tenerife (Canary Islands): relationship to vegetation zones and environmental factors.

Knowledge about fungal diversity scaling relationships relative to that of plants is important to understand ecosystem functioning. Tenerife Island, a natural laboratory to study terrestrial biodiversity, is represented by six different vegetation zones characterized by specific abiotic conditions and plant communities with a high proportion of endemic plants. Little is known about the biodiversity of associated fungi. To understand the relationship between plant and fungal communities, we analysed soil/rhizosphere fungi from all vegetation zones. From 12 sampling points dispersed on the whole island, molecular analysis of fungal communities was determined by single-strand conformation polymorphism (SSCP) analysis using universal and specific primers for Trichoderma. The highly diverse fungal communities were mainly characterized by ectomycorrhiza-forming Basidiomycota and a high proportion of yet-unidentified species. Besides, Trichoderma-specific SSCP resulted in low diversity of mainly cosmopolitan species, for example Hypocrea lixii/T. harzianum. The dominance of T. harzianum was confirmed by cultivation. All Trichoderma isolates show an extraordinarily high antagonistic potential towards different groups of plant pathogens, supporting the hypothesis of extensive colonization by highly competitive Trichoderma species from the continent. In contrast, biodiversity patterns of the whole fungal and plant communities follow the same ecological rules. Furthermore, a high statistical correlation between fungal communities and the main environmental factors, temperature and precipitation, was found.

Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/Trichoderma.

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.

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.

Fungal genus Hypocrea/Trichoderma: from barcodes to biodiversity.

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.

Formation of atroviridin by Hypocrea atroviridis is conidiation associated and positively regulated by blue light and the G protein GNA3.

Species of the mycoparasitic fungal genus Hypocrea/Trichoderma are prominent producers of peptaibols, a class of small linear peptides of fungal origin. Some of these peptaibols have been shown to act synergistically with cell-wall-degrading enzymes in the inhibition of the growth of other fungi in vitro and in vivo. Here we present the structure of the Hypocrea atroviridis peptaibol synthetase gene (pbs1), deduced from the genome sequence of H. atroviridis. It consists of 19 typical peptide synthetase modules with the required additional modifying domains at the N and C termini. Phylogenetic and similarity analyses of the individual amino acid-activating modules is consistent with its ability to synthesize atroviridins. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of surface-grown cultures of H. atroviridis showed that no peptaibols were formed during vegetative growth, but a microheterogenous mixture of atroviridins accumulated when the colonies started to sporulate. This correlation between sporulation and atroviridin formation was shown to be independent of the pathway inducing sporulation (i.e., light, mechanical injury and carbon starvation, respectively). Atroviridin formation was dependent on the function of the two blue light regulators, BLR1 and BLR2, under some but not all conditions of sporulation and was repressed in a pkr1 (regulatory subunit of protein kinase A) antisense strain with constitutively active protein kinase A. Conversely, however, loss of function of the Galpha-protein GNA3, which is a negative regulator of sporulation and leads to a hypersporulating phenotype, fully impairs atroviridin formation. Our data show that formation of atroviridin by H. atroviridis occurs in a sporulation-associated manner but is uncoupled from it at the stage of GNA3.

Application of DNA bar codes for screening of industrially important fungi: the haplotype of Trichoderma harzianum sensu stricto indicates superior chitinase formation.

Selection of suitable strains for biotechnological purposes is frequently a random process supported by high-throughput methods. Using chitinase production by Hypocrea lixii/Trichoderma harzianum as a model, we tested whether fungal strains with superior enzyme formation may be diagnosed by DNA bar codes. We analyzed sequences of two phylogenetic marker loci, internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA-encoding gene cluster and the large intron of the elongation factor 1-alpha gene, tef1, from 50 isolates of H. lixii/T. harzianum, which were also tested to determine their ability to produce chitinases in solid-state fermentation (SSF). Statistically supported superior chitinase production was obtained for strains carrying one of the observed ITS1 and ITS2 and tef1 alleles corresponding to an allele of T. harzianum type strain CBS 226.95. A tef1-based DNA bar code tool, TrichoCHIT, for rapid identification of these strains was developed. The geographic origin of the strains was irrelevant for chitinase production. The improved chitinase production by strains containing this haplotype was not due to better growth on N-acetyl-beta-D-glucosamine or glucosamine. Isoenzyme electrophoresis showed that neither the isoenzyme profile of N-acetyl-beta-glucosaminidases or the endochitinases nor the intensity of staining of individual chitinase bands correlated with total chitinase in the culture filtrate. The superior chitinase producers did not exhibit similarly increased cellulase formation. Biolog Phenotype MicroArray analysis identified lack of N-acetyl-beta-D-mannosamine utilization as a specific trait of strains with the chitinase-overproducing haplotype. This observation was used to develop a plate screening assay for rapid microbiological identification of the strains. The data illustrate that desired industrial properties may be an attribute of certain populations within a species, and screening procedures should thus include a balanced mixture of all genotypes of a given species.

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).

Facts and challenges in the understanding of the biosynthesis of peptaibols by Trichoderma.

Species of the mitosporic filamentous fungal genus Trichoderma are prominent producers of both short (7-11 residues) and long (18-20 residues) peptaibols and peptaibiotics, which are thought to be involved in their interaction with other living systems. Numerous reviews are available regarding biodiversity, structure, and mode of action of these peptide derivatives, but little emphasis has been paid to the physiology and genetics of their formation. In this review article, we used the recent knowledge on biosynthesis and production of these components to speculate on some of the unknown points. We also highlight areas where further research is most urgently needed.