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).     

木霉属中国新纪录种Trichoderma pleuroticola和T. pleurotum

【目的】对蔬菜大棚土壤中和阿魏菇腐烂的菌盖上分离的两株木霉菌进行分类鉴定。【方法】结合形态学分类特征和ITS序列分析的方法进行鉴定。【结果】从蔬菜大棚的土壤中和阿魏菇腐烂的菌盖上分离的两株木霉菌分别为Trichoderma pleuroticola和T.pleurotum。T.pleuroticola的形态特征与T.harzianum相似,但其分生孢子显著大于T.harzianum的分生孢子,且在PDA上产生黑褐色的色素以及黄色的结晶物。T.pleurotum典型特征是分生孢子梗单生,有时匍匐,分枝散生,初级分枝和分生孢子梗顶端聚生,类似粘帚霉。【结论】分离的两株木霉分别是T.pleuroticola和T.pleurotum,为木霉菌中国新纪录种。     

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/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.     

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.     

Hypocrea atroviridis sp. nov., the teleomorph of Trichoderma atroviride

A new species, Hypocrea atroviridis, is described for the teleomorph of Trichoderma atroviride. Based on sequences of ITS-1, 5.8S, and ITS-2 regions of the rDNA complex and translation-elongation factor (EF-1α), T. atroviride and H. atroviridis form a well-supported clade within Trichoderma sect. Trichoderma. The conserved anamorphic phenotype of T. atroviride, observed for both conidial and ascospore derived cultures, was only found within that clade. In contrast, the teleomorph phenotype of H. atroviridis was morphologically indistinguishable from H. rufa, the teleomorph of T. viride. This Hypocrea phenotype may, therefore, be considered to be plesiomorphic within Trichoderma sect. Trichoderma, suggesting that genes controlling the expression of the teleomorph and anamorph evolve at different rates and that the genes controlling expression of the teleomorph are more conserved than are those controlling the expression of the anamorph.     

Isolate-specific conidiation in Trichoderma in response to different nitrogen sources

A characteristic feature of Trichoderma is the production of concentric rings of conidia in response to alternating light/dark conditions and a single ring of conidia in response to a single burst of light. In this study, conidiation was investigated in four biocontrol isolates (T. hamatum, T. atroviride, T. asperellum, T. virens) and one isolate from the mushroom pathogen species, T. pleuroticola. All five isolates produced concentric conidial rings under alternating light/dark conditions on potato-dextrose agar (PDA), however, in response to a 15min burst of blue light, only T. asperellum and T. virens produced a clearly defined conidial ring. Both T. pleuroticola and T. hamatum photoconidiated in a disk-like fashion and T.?atroviride produced a broken ring with a partially filled in appearance. In the presence of primary nitrogen, T. asperellum and T. pleuroticola conidiated in a disk, whereas, when grown in the presence of secondary nitrogen, a ring of conidia was produced. Primary nitrogen promoted photoconidiation and competency to conidiate in response to light appeared dependent on the nitrogen catabolite repression state of the cell. Mycelial injury was also investigated in the same five isolates of Trichoderma on PDA and under different nitrogen statuses. For the first time, we report that conidiation in response to injury is differentially regulated in different isolates/species of Trichoderma.     

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.     

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.     

Trichoderma biodiversity in China

In the present study, we made further investigation into the diversity of Trichoderma in China than previous ones utilizing comprehensive approaches of morphological microscopic observation and phylogenetic analysis by detecting molecular markers. One thousand nine hundred ten Trichoderma strains were isolated from soil or other materials in China: East (Anhui, Fujian, Jiangsu, Jiangxi, Shandong, Zhejiang province and Shanghai municipality), South-West (Guizhou, Qinghai, Shanxi, Sichuan and Yunnan province, Tibet Autonomous Region and Chongqing municipality), South-East (Guangdong, Guangxi, Hainan province), and Middle China (Henan, Hubei and Hunan province). Representative isolates were verified at the species level by morphological characters and the oligonucleotide barcode program TrichoOKey v.10 and the custom BLAST server TrichoBLAST, using sequence of the ITS 1 and 2 region of the rDNA cluster and partial sequences of translation elongation factor 1-alpha(tef1-α). A total of 23 Trichoderma species were identified : T.asperellum, T.atrioviride, T.aureovriride, T.brevicompactum, T.citrioviride, T.erinaceum, T.gamsii, T.hamatum, T.harzianum (H.1ixii), T.intricatum, T.koningii (H.koningii), T.koningiopsis, T.longibranchiatum, T.pleuroticola, T.reeseii (H.jecorina), T.sinensis, T.spirale, T.stromaticum, T.tomentosum, T.velutinum, T.vermipilum, T.virens (H.virens), T.viride. Among them, 3 species: T.intricatum, T.stromaticum, T.vermipilum were first reported in China; T.harzianum (H,1ixii) was the most widely distributed species in China. This study further shows that, the highest biodiversity of Trichoderma population appeared in South-West China.     

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.     

Direct identification of hydrophobins and their processing in Trichoderma using intact-cell MALDI-TOF MS

Intact-cell MS (ICMS) was applied for the direct detection of hydrophobins in various species and strains of Hypocrea/Trichoderma. In both mycelia and spores, dominating peaks were identified as hydrophobins by detecting mass shifts of 8 Da of reduced and unreduced forms, the analysis of knockout mutants, and comparison with protein databases. Strain-specific processing was observed in the case of Hypocrea jecorina (anamorph Trichoderma reesei). An analysis of 32 strains comprising 29 different species of Trichoderma and Hypocrea showed hydrophobin patterns that were specific at both at the species and isolate (subspecies) levels. The method therefore permits rapid and direct detection of hydrophobin class II compositions and may also provide a means to identify Trichoderma (and other fungal) species and strains from microgram amounts of biomass without prior cultivation.     

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 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.     

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。     

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.     

Ambient pH intrinsically influences Trichoderma conidiation and colony morphology

Conidiation in Trichoderma has been demonstrated to be favoured by a low ambient pH and more recently PacC (Pac1) mediated pH-regulation has been implicated in the control of conidiation. In this study, ambient pH effects on conidiation were investigated in three isolates (Trichoderma hamatum, Trichoderma atroviride and Trichoderma pleuroticola) exposed to a single blue-light burst or to mycelial injury. Disks of conidiation were observed for T. atroviride in response to a single light exposure, which clearly demonstrates that all cells are potentially competent for photoconidiation. Previous studies have suggested T. hamatum does not conidiate in response to mycelial injury, however, in this study a clear injury response was observed from pH 2.8 to 3.2. T. pleuroticola displayed three distinct pH-dependent colony morphologies from pH 2.8 to 5.2. Conidiation was strictly low pH-dependent on buffered media and observed at all pH values on unbuffered media. The dependence of the conidial phenotype on the buffering state of the medium rather than the pH per se, was unexpected as it has been suggested that conidiation is PacC regulated. Conversely, excretion of an anthraquinone was strictly pH-dependent regardless of the buffering state. These studies highlight the complexity of ambient pH effects on Trichoderma spp. and demonstrate a need to widen the scope of research to multiple species.     

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.