木霉属Trichoderma组和Pachybasium组的分子系统学研究

对来源不同的木霉及其有性型Longibrachiatum组、Trichoderma 组和Pachybasium组的81个菌株进行了ITS序列测定,并对ITS1序列用PHYLIP程序包中的DNAPARS程序进行系统发育分析。结果表明Trichoderma 组和Pachybasium组的所有菌株可分成两个群(A,B),B群进一步分为4个分支(B1,B2,B3,B4);A群由Trichoderma 组的H. aureoviridis和未鉴定到种的3个Hypocrea菌株构成;B1,B2,B4群均由Pachybasium组菌株构成;B3群由Pachybasium组的T. hamatum、T. strigosum和Trichoderma 组的T. asperellum、T. atroviride、T. koningii、T. viride和Hypocrea菌株T261构成。2个组相互交叉,组间没有明确的区分,进一步证明Pachybasium组是多系的。建议将Trichoderma 组中的T. viride aggr.、T. atroviride、T. koningii归并入Pachybasium组,对Trichoderma 组重新定义。     

Taxonomy and phylogenetic relationships of two species of Hypocrea with Trichoderma anamorphs

Hypocrea patella is reevaluated. Its Trichoderma anamorph is described and the phylogenetic position of the species is determined through sequences of the ITS regions of rDNA. It is sister to a clade that includes Trichoderma longibrachiatum/H. schweinitzii. Hypocrea patella f. tropica is accepted for a Costa Rican collection. Hypocrea neorufa and its Trichoderma anamorph are described. Its phylogenetic position is determined by sequences of the ITS region of rDNA and the protein-coding translation-elongation factor (EF-1α). It is derived from within a clade that includes T. viride/H. rufa, T. atroviride/H. atroviridis, T. koningii/H. koningii and T. asperellum. Deceased August 2002.     

Blue pigment in Hypocrea caerulescens sp. nov. and two additional new species in sect. Trichoderma

Three new species of Hypocrea/Trichoderma sect. Trichoderma (Hypocreaceae, Hypocreales, Ascomycota, Fungi) are described from recent collections in southern Europe and the Canary Islands. They have been characterized by morphological and molecular methods, including microscopic examination of the teleomorph in thin sections, the anamorph, growth rate experiments and phylogenetic analyses based on a part of the translation elongation factor 1-alpha encoding gene (tef1) containing the two last introns and a part of the rpb2 gene, encoding the second largest RNA polymerase subunit. Analyses involving tef1 did not unequivocally resolve the sister clade relationship of Hypocrea caerulescens relative to the Koningii and Viride clades, while analyses based on rpb2 clearly suggest a close relationship with the former, although the phenotype of H. caerulescens is similar to H. viridescens, particularly by its warted conidia and a coconut-like odor in CMD culture. Hypocrea hispanica and T. samuelsii however are clearly related to the Viride clade by both phylogenetic markers, despite their morphological similarity to H. koningii and its relatives. An apparently specific blue pigment is formed in CMD cultures by Hypocrea caerulescens but could not be obtained by extraction with organic solvents.     

Hypocrea voglmayrii sp. nov. from the Austrian Alps represents a new phylogenetic clade in Hypocrea/Trichoderma

The holomorph of the new species Hypocrea voglmayrii (Hypocreales, Ascomycota, Fungi) is described by a combined approach, using morphology of the teleomorph, morphology of the anamorph, culture studies and phylogenetic analyses of ITS1 and 2, ech42 and rpb2 gene sequences. Its anamorph Trichoderma voglmayrii is described as a new anamorph species. Unlike most other species of Hypocrea the teleomorph of H. voglmayrii occurs on dry standing trunks and exhibits well defined black ostioles. Although exclusively collected at higher altitudes, this species grows at 35 C in culture. Hypocrea voglmayrii develops pale yellowish to greenish conidia, a yellowish pigment and a coconut-like odor on CMD. Phylogenetically, H. voglmayrii forms a distinct, isolated branch between the section Trichoderma and the H. pachybasioides clade but does not associate with any of these clades in different gene trees.     

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 ( TrichO KEY), sequence similarity analysis ( Tricho blast ) 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.     

Hypocrea crystalligena sp. nov., a common European species with a white-spored Trichoderma anamorph

The new species Hypocrea crystalligena (Hypocreales, Ascomycota, Fungi) is described as a holomorph and characterized based on an integrated phenotypic and phylogenetic approach, using teleomorph and anamorph morphologies, culture studies and analyses of phylogenetic markers including internal transcribed spacer 1 and 2 (ITS1 and 2), two last introns of the translation elongation factor 1-alpha encoding gene (tef1), and a portion of the rpb2 gene, encoding the second largest RNA polymerase subunit. Stromata of H. crystalligena show similarities with those of species from Trichoderma sect. Trichoderma but differ in several respects, including color, presence of white crystals on the surface and small ascospores. Colonies on CMD appear distinct, form colorless to white crystals on isolation, a yellowish to brown pigment and an anamorph with hyaline conidia exhibiting verticillium-like to gliocladium-like structural elements. ITS1 and 2 sequences exhibit all genus-specific features but also contain several unique hallmarks permitting development of a species-diagnostic barcode. Based on the analyses of partial rpb2 and tef1 sequences, H. crystalligena constitutes a separate evolutionary lineage with H. megalocitrina and H. psychrophila as its nearest neighbors. All these species form one phylogenetic clade with the H. pulvinata/H. citrina node.     

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.     

Hypocrea britdaniae and H. foliicola: two remarkable new European species

Two new species of Hypocrea are added here to the European funga. Hypocrea britdaniae, a fungus with unknown anamorph and large, conspicuous stromata resembling basidiomata of a corticiaceous fungus, is a sister species to the Longibrachiatum clade, while H. foliicola, a leaf-dwelling species that forms pulvinate stromata, is recognized as an additional member of the pachybasium core group. Hypocrea foliicola sporulates in culture in a reduced verticillium-like manner, while it produces a white, typical pachybasium-like anamorph in nature. Ecologically H. foliicola is remarkable in inhabiting leaves, a substrate rarely recorded for Hypocrea. All relevant morphological teleomorphic and anamorphic traits are given. The phylogenetic placement of the new species within Hypocrea/Trichoderma was determined with combined analyses of rpb2 and tef1 exon sequences.     

Hypocrea lixii, the teleomorph of Trichoderma harzianum

Cultures derived from ascospores of Hypocrea lixii (= H. nigricans, H. lentiformis) produced the morphological species Trichoderma harzianum in pure culture. Trichoderma harzianum, the most commonly found species of the genus, is also one of the most species frequently used in biocontrol of plant pathogens. It has not been connected previously to a teleomorph. The connection was confirmed by DNA sequence analysis. Similar to the anamorph, the teleomorph collections have a wide geographic distribution. Described in the 19^th century, Hypocrea lixii is epitypified by a collection from Thailand.     

Phylogeny of the genus trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA cluster

Sequences of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA were used to determine the phylogenetic relationships of species of Trichoderma sect. Pachybasium. To this end, 85 strains-including all the available ex-type strains-were analyzed. Parsimony analysis demonstrated that the section is nonmonophyletic, distributing the 85 strains among three main groups that were supported by bootstrap values. Group A comprises two clades (A1 and A2), with A1 including T. polysporum, T. piluliferum, and T. minutisporum, while A2 included T. hamatum, T. pubescens, and T. strigosum in addition to species previously included in sect. Trichoderma (i.e., T. viride, T. atroviride, and T. koningii). The ex-type strain of T. fasciculatum formed a separate branch basal to clade A. Clade B contained the sect. Pachybasium members T. harzianum, T. fertile, T. croceum, T. longipile, T. strictipile, T. tomentosum, T. oblongisporum, T. flavofuscum, T. spirale, and the anamorphs of Hypocrea semiorbis and H. cf. gelatinosa. Sequence differences among clades A1, A2, and B were in the same order of magnitude as between each of them and T. longibrachiatum, which was used as an outgroup in these analyses. Sequence differences within clades A1, A2, and B were considerably smaller: in some cases (i.e., T. virens and T. flavofuscum; T. strictipile and H. cf. gelatinosa), the ITS1-sequences were identical, suggesting conspecifity. In other cases (e.g., T. crassum and T. longipile; T. harzianum, T. inhamatum, T. croceum, T. fertile, and H. semiorbis; T. hamatum and T. pubescens; and T. viride, T. atroviride, and T. koningii) differences were in the range of 1-3 nt only, suggesting a very close phylogenetic relationship. The sequence of a previously described aggressive mushroom competitor group of T. harzianum strains (Th2) was strikingly different from that of the ex-type strain of T. harzianum and closely related species and is likely to be a separate species. Copyright 1998 Academic Press.     

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.     

Hypocrea/Trichoderma species with pachybasium-like conidiophores: teleomorphs for T. minutisporum and T. polysporum and their newly discovered relatives

We describe or redescribe species of Hypocrea/Trichoderma (Ascomycetes, Hypocreales) having hyaline ascospores and pachybasium-like conidiophores. Teleomorphs are reported for Trichoderma minutisporum (Hypocrea minutispora sp. nov.) and T. polysporum (H. pachybasioides). Hypocrea pilulifera/T. piluliferum is redescribed. Trichoderma croceum is synonymized with T. polysporum. The new species H. parapilulifera, H. stellata and H. lacuwombatensis are described. All of these species fall within the morphological concept of Trichoderma sect. Pachybasium and within the phylogenetic group pachybasium B5 of Kullnig-Gradinger et al (2002). Parsimony analysis of nucleotide sequences from three unlinked loci-ITS1 and 2, endochitinase (ech42) and translation elongation factor 1-alpha (tef1)-detects two distinct phylogenetic lineages within the group pachybasium B5. One comprises H. pachybasioides/T. polysporum, H. pilulifera/T. piluliferum, H. parapilulifera and H. stellata; this group, the "polysporum" lineage, is characterized by having conidia that are white in mass and is the only lineage within Hypocrea characterized by such conidia. The second group includes the green conidial T. minutisporum and H. lacuwombatensis. The partition homogeneity test reveals significant recombination within the "polysporum" lineage but not within the "minutisporum" lineage.     

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.     

A morphological and molecular perspective of Trichoderma viride: is it one or two species?

Trichoderma (Ascomycetes, Hypocreales) strains that have warted conidia are traditionally identified as T. viride, the type species of Trichoderma. However, two morphologically distinct types of conidial warts (I and II) have been found. Because each type corresponds to a unique mitochondrial DNA pattern, it has been questioned whether T. viride comprises more than one species. Combined molecular data (sequences of the internal transcribed spacer 1 [ITS-1] and ITS-2 regions and of part of the 28S rRNA gene along with results of restriction fragment length polymorphism analysis of the endochitinase gene and PCR fingerprinting), morphology, physiology, and colony characteristics distinguish type I and type II as different species. Type I corresponds to "true" T. viride, the anamorph of Hypocrea rufa. Type II represents a new species, T. asperellum, which is, in terms of molecular characteristics, close to the neotype of T. hamatum.     

Hypocrea peltata: a mycological Dr Jekyll and Mr Hyde?

Hypocrea peltata (Pezizomycotina, Hypocreales, Hypocreaceae) is a common, widespread essentially subtropical species, with an uncharacteristically large stroma and asci containing four large and four small bicellular ascospores. Its only anamorph consists of indehiscent aleuriospores; it does not form a Trichoderma anamorph, which is typical of most Trichoderma/Hypocrea species. Hypocrea peltata grows very well at 37 C. The large stromata and failure to form a Trichoderma anamorph could lead one to doubt its generic placement. However sequences of the internal transcribed spacer region (ITS), 28S nuclear large subunit (LSU) of rDNA and RNA polymerase subunit II (rpb2) regions indicate that it represents a unique lineage within Trichoderma/Hypocrea. ITS and rbp2 sequences derived from cultures of H. peltata are identical to the "unidentified Hypocreaceae" reported in the literature as being isolated from lung of a patient with non-fatal pulmonary fibrosis.     

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.     

丝状真菌红褐肉座菌(Hypocrea jecorina)纤维素酶基因的转录调控研究进展

红褐肉座菌(Hypocrea jecorina,即Trichoderma reesei的有性型)是工业上重要的纤维素酶生产菌株,也是用于研究纤维素酶和半纤维素酶基因转录调控机制的模式菌株。在诱导物存在的条件下,H.jecorina可以迅速启动这些糖苷水解酶基因的转录表达,但不同的诱导物对纤维素酶和半纤维素酶基因的诱导表达模式存在一定差异。目前对不溶性诱导物如结晶纤维素如何诱导这些基因的起始转录问题有3种假设;并且已发现某些参与调控纤维素酶基因转录的正调控因子(Xyr1、Ace2、Hap2/3/5)和负调控因子(Ace1、Cre1),这些调控因子可在纤维素酶基因启动子上结合且彼此间可能发生相互作用。本文系统综述了红褐肉座菌纤维素酶基因转录表达调控中的关键因素及其相互作用的相关研究进展。