Christian P. Kubicek

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.

Regulation of citric acid production by oxygen: Effect of dissolved oxygen tension on adenylate levels and respiration in Aspergillus niger

SummaryThe mechanism of the control of citric acid accumulation by oxygen was investigated by means of pilot plant fermentation using Aspergillus niger. The critical dissolved oxygen tension (DOT) for oxygen uptake of this fungus was about 18–21 and 23–26 mbar for trophophase and idiophase, respectively. Minimal DOT for citric acid production was about 25 mbar. Citric acid production increased steadily between 40–150 mbar. Short time changes in the DOT produced immediate, irreversible changes in the rate of product formation. Adenine nucleotides paralleled growth but showed no evidence for control function in the oxygen effect on citric acid fermentation. A branched respiratory system was identified by experiments using specific inhibitors (antimycin, cyanide, azide, rotenone, amytal and salicylhydroxamic acid). Growth was sensitive towards inhibitors of the standard respiratory chain, but only slightly sensitive towards salicylhydroxamic acid (SHAM). Citric acid synthesis was highly sensitive towards SHAM during trophophase, but sensitive towards antimycine during idiophase. Interruptions in aeration cause an impairment of the SHAM sensitive oxidase during trophophase, and of the antimycin sensitive oxidase during idiophase.

Revision of Trichoderma sect. Longibrachiatum including related teleomorphs based on analysis of ribosomal DNA internal transcribed spacer sequences

Variation within the internal transcribed spacer (ITS-1 and ITS-2) regions of ribosomal DNA of 103 strains was studied to examine relationships within Trichoderma sect. Longibrachiatum and related teleomorphs, Hypocrea schweinitzii and H. jecorina. The four species, T longibrachiatum, T. pseudokon- ingii, T parceramosum and T citrinoviride, that were originally assigned by morphological criteria to this section were recognized by sequence analysis. Trich- oderma reesei, which was previously considered to be synonymous with T longibrachiatum, and T saturnis- porum which was placed originally in sect. Saturnis- porum because of its warted conidia, are assigned to the section. ITS sequences of T ghanense, originally assigned to sect. Saturnisporum, could not be distin- guished from the sequence of T parceramosum, in- dicating that the two may be synonymous. Trichoder- ma todica, an unpublished name based on the an-

The Nag1 N -acetylglucosaminidase of Trichoderma atroviride is essential for chitinase induction by chitin and of major relevance to biocontrol

Abstract The nag1 gene of the mycoparasitic fungus Trichoderma atroviride encodes a 73-kDa N-acetyl-β-d-glucosaminidase, which is secreted into the medium and partially bound to the cell wall. To elucidate the role of this enzyme in chitinase induction and biocontrol, a nag1-disruption mutant was prepared. It displayed only 4% of the original N-acetyl-β-d-glucosaminidase activity, indicating that the nag1 gene product accounts for the majority of this activity in T. atroviride. The nag1-disruption strain was indistinguishable from the parent strain in growth and morphology, but exhibited delayed autolysis. Northern analysis showed that colloidal chitin disruption does not induce ech42 gene transcription in the nag1-disruption strain. Enzyme activities capable of hydrolysing p-nitrophenyl-N,N′-diacetylchitobioside and p-nitrophenyl-N,N′-diacetylchitotriose were also absent from the nag1-disruption strain under the same conditions. Retransformation of the T. atroviride nag1-disruption strain with the nag1 gene essentially led to the parent-type behaviour in all these experiments. However, addition of N-acetyl-β-d-glucosaminidase to the medium of the nag1-disruption strain did not rescue the mutant phenotype. The disruption-nag1 strain showed 30% reduced ability to protect beans against infection by Rhizoctonia solani and Sclerotinia sclerotiorum. The data indicate that nag1 is essential for triggering chitinase gene expression in T. atroviride and that its functional impairment reduces biocontrol by T. atroviride by a significant extent.

Regulation of chitinase 33 (chit33) gene expression in Trichoderma harzianum.

Abstract We investigated the regulation of chit33 expression in Trichoderma harzianum CECT 2413. This gene encodes the Chit33 endochitinase, which is a major component of the fungus chitinolytic enzyme system and is important for biocontrol. To this end, both Northern analysis and reporter gene fusions of a 1.4-kb fragment of the 5′-upstream sequences of chit33 to the Aspergillus nigergoxA gene (encoding glucose oxidase) and the Aquorea victoria green fluorescent protein were used. Northern analysis and data obtained with the reporter systems were compatible, thus showing that the 1.4-kb fragment bears all necessary information for the regulation of chit33 gene expression. chit33 is weakly expressed during growth on chitin and Rhizoctonia solani cell walls. The addition of N-acetylglucosamine transiently induced chit33 expression in resting cells of the fungus. The addition of either glucose or glycerol prevented induction of chit33 gene expression by chitin or cell walls. Incubation of T. harzianum in the presence of low concentrations (0.1%, w/v) of glucose and high concentrations (38u2009mM) of ammonium sulfate, or in the presence of high concentrations (1%, w/v) of glucose and low concentrations (0.38u2009mM) of ammonium sulfate also stimulated chit33-mRNA accumulation, although to a lower degree than induction by N-acetylglucosamine. Transfer of T. harzianum cultures to either 40u2009°C or 4u2009°C initiated a very rapid expression of chit33 in the absence of an inducer, yet only at very low levels (5%) of the induced control. Confrontation experiments, using the gfp gene as a reporter and R. solani as a host, showed that chit33 is expressed only during but not before the stage of overgrowth on R. solani. These data show that Chit33 is an enzyme involved in mycoparasitism; and its formation is controlled by induction, by either carbon or nitrogen starvation and, to a low degree, also under conditions of temperature stress.

Nucleosome transactions on the Hypocrea jecorina ( Trichoderma reesei) cellulase promoter cbh2 associated with cellulase induction

The 5′ regulatory region of the cbh2 gene of Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for induction of cbh2 gene expression by sophorose and cellulose. The CAE consists of two motifs, a CCAAT box on the template strand and a GTAATA box on the coding strand, which cooperate during induction. Northern analyses of cbh2 gene expression has revealed an absolute dependence on induction, but no direct effect of Cre1-mediated carbon catabolite repression. Investigation of the chromatin structure in the wild-type strain showed that, under repressing conditions, there is a nucleosome free region (nfr) around the CAE, which is flanked by strictly positioned nucleosomes. Induction results in a loss of positioning of nucleosomes −1 and −2 downstream of the CAE, thus making the TATA box accessible. Simultaneous mutation of both motifs of the CAE, or of the CCAAT-box alone, also leads to shifting of nucleosome −1, which normally covers the TATA-box under repressing conditions, whereas mutation of the GTAATA element results in a narrowing of the nfr, indicating that the proteins that bind to both motifs in the CAE interact with chromatin, although in different ways. A cellulase-negative mutant strain, which has previously been shown to be altered in protein binding to the CAE, still displayed the induction-specific changes in nucleosome structure, indicating that none of the proteins that directly interact with CAE are affected, and that nucleosome rearrangement and induction of cbh2 expression are uncoupled. Interestingly, the carbon catabolite repressor Cre1 is essential for strict nucleosome positioning in the 5′ regulatory sequences of cbh2 under all of the conditions tested, and induction can occur in a promoter that lacks positioned nucleosomes. These data suggest that Cre1, the Hap2/3/5 complex and the GTAATA-binding protein are all involved in nucleosome assembly on the cbh2 promoter, and that the latter two respond to inducing conditions by repositioning nucleosome −1.

Phosphorylation positively regulates DNA binding of the carbon catabolite repressor Cre1 of Hypocrea jecorina (Trichoderma reesei).

Cre1 of the ascomycete Hypocrea jecorina is a Cys2His2 zinc finger DNA-binding protein functioning as regulator for carbon catabolite repression. It represents the functional equivalent of yeast Mig1, known to be negatively regulated by the Snf1-kinase at the nuclear import level. We demonstrate that Cre1 is also a phosphoprotein, and identify Ser241 within an acidic protein region as phosphorylation target. In contrast to Mig1 phosphorylation is required for DNA binding of Cre1. A S241E mutation mimics phosphorylation, whereas a S241A mutant protein shows phosphorylation-independent DNA binding activity, suggesting that phosphorylation is required to release Cre1 from an inactive conformation involving unphosphorylated Ser241. Retransformation of a H. jecorina cre1-non functional mutant with Cre1-S241A leads to permanent carbon catabolite repression in cellobiohydrolase I expression. Contrary to Mig1, the amino acid sequence surrounding Ser241 (HSNDEDD) suggests that phosphorylation may occur by a casein kinase II-like protein. This is supported by a mutation of E244V leading to loss of phosphorylation, loss of DNA binding, and gain of carbon catabolite derepression. Our results imply that the regulation of carbon catabolite repression at the level of DNA binding strongly differs between Saccharomyces cerevisiae andH. jecorina.

Biogeography and phenotypic variation in Trichoderma sect. Longibrachiatum and associated Hypocrea species

Random amplification of polymorphic DNA (RAPD), using both fingerprinting (M13, [GTG]5, [GACA]4) and random [V5] primers, has been used to analyse 145 isolates characteristic of Trichoderma sect. Longibrachiatum and Hypocrea species with anamorphs referable to that section, and to identify strains with named species of sect. Longibrachiatum. To this end, ex-type strains of T. longibrachiatum, T. pseudokoningii, T. citrinoviride, T. parceramosum and T. reesei were analysed, and the similarity coefficients of RAPD characters used as a criterion for the alignment of strains to individual species. The ex-type strains of T. saturnisporum and T. ghanense exhibited the same interspecific similarity index as the other ex-type strains of Trichoderma sect. Longibrachiatum, and clustered well within it and were therefore included into this section. 103 isolates were identified as members of Trichoderma sect. Longibrachiatum and further investigated. Most of the anamorphic isolates could be aligned with T. citrinoviride or T. longibrachiatum, whereas only a few strains of T. saturnisporum and T. parceramosum were found. No naturally occurring anamorphs with homology to the ex-type strains of T. reesei and T. pseudokoningii were found. While T. longibrachiatum and T. citrinoviride overlapped through much of their geographic ranges, the former was present in Africa and India but not in southeast Asia, while the reverse was true of the latter. The African strains of T. longibrachiatum were equally distant from the ex-type strains of T. longibrachiatum, T. parceramosum and T. saturnisporum. The type strain of T. ghanense was identified as belonging to this group. T. pseudokoningii, the anamorph of Hypocrea schweinitzii, was found exclusively in eastern Australia and New Zealand. Attempts to identify potential teleomorphs of the other anamorphs revealed another subgroup of H. schweinitzii, occurring in temperate climates in Europe and U.S.A., as homologous to T. citrinoviride. No Hypocrea strains could be identified as teleomorphs of T. longibrachiatum, T. parceramosum or T. saturnisporum.

Molecular identification of Trichoderma species from Russia, Siberia and the Himalaya

About 35 Trichoderma species are currently recognised on the basis of morphological and molecular characters. Besides the role of a few of these species in biotechnology, several seem to play prominent roles in soil ecosystems. With a goal of investigating global biodiversity in Trichoderma , we report on the occurrence of Trichoderma spp. in Russia (Moscow and Ural areas), Siberia (Krasnoyarsk area) and the Himalayan mountains — areas from which no Trichoderma isolates are so far available. The ITS 1 and 2 sequence of the rDNA cluster of the 75 isolates obtained was compared with that of ex-type strains and taxonomically established isolates of Trichoderma . Thirty-nine isolates were positively identified as T. atroviride, T. virens, T. hamatum, T. asperellum, T. koningii and T. oblongisporum . A further 26 isolates yielded six closely related ITS1/2 sequence types, which are highly similar yet different from the ex-(neo)type strains of T. harzianum and T. inhamatum . Some of these genotypes (i.e. 1 and 2a) occurred only in Russia/Siberia, whereas others (2b, 3, 4 and 5) were found only in the Himalayas. RAPD analysis was consistent with these genotypes, and revealed genetic homogeneity even between strains from widely separated areas. Parsimony analysis placed these five genotypes, together with T. harzianum, T. inhamatum and the mushroom-aggressive T. harzianum ‘biotype 2’ in a large, unresolved ‘ harzianum ’ clade. Ten isolates were not safely alignable within known species, and five of them may be undescribed taxa: one isolate from 2700 m elevation in the Himalayas, which clustered in parsimony analysis at a basal position in section Longibrachiatum ; and four isolates, displaying two closely related sequence types, forming a separate clade with T. stromaticum . The five remaining isolates also exhibited three unique ITS1 and 2 sequence patterns, but parsimony analysis placed them into the unresolved ‘ harzianum ’ clade, and their relationship to T. harzianum is thus unclear. The study shows that molecular screening of uninvestigated geographic areas can lead to the identification of isolates with new ITS1 and ITS2 sequence patterns, some of which may be new taxa. It also reveals that T. harzianum is at present the genetically most diverse member of the genus.

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.