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Objective: Trichoderma species are found in soil and in association with plants. They can act directly or indirectly in the biological control of plant diseases and in the promotion of plant growth, being among the most used fungi in the formulation of bioproducts applied to agricultural systems. The main objective of this study was to characterize at a first-tier level a collection of 67 Trichoderma isolates from various tropical sources, based solely on sequencing of the internal transcribed spacer (ITS) region of the rRNA genes. Our goal was to provide a preliminary idea of the baseline diversity in this collection, to combine this information later with an array of other isolate-specific physiological data. This study provides a required knowledge at molecular level for assessment of this germplasm potential as a source of biotechnological products for beneficial effects in plants., Results: Sequencing of the ITS region showed that the 67 Trichoderma isolates belonged in 11 species: T. asperellum, T. atroviride, T. brevicompactum, T. harzianum, T. koningiopsis, T. longibrachiatum, T. pleuroticola, T. reesei, T. spirale, T. stromaticum and T. virens. A total of 40.3% of the isolates were very closely related to each other and similar to T. harzianum. The baseline genetic diversity found indicates that the collection has different genotypes, which can be exploited further as a source of bioproducts, aiming at providing beneficial effects to plants of interest to cope with biotic and abiotic stresses.

Trichoderma is one of the most exploited biocontrol agent for the management of plant diseases. Twenty strains of Trichoderma (six of T. harzianum, four of T. viride, three of T. virens, three of T. koningii, each one of T. hamatum, T. reesei, T. parceramosum and Trichoderma spp.) subjected to in vitro antagonism up to 12days after inoculation against Sclerotium rolfsii Sacc. causing stem rot in groundnut. A new concept was developed to determine inhibition coefficient representing pathogen biology and biocontrol related biophysical variables. Results explained differential inhibition coefficient of test pathogen by Trichoderma antagonists. The inhibition coefficient of test pathogen was examined highest (91.13%) by T. virens NBAII Tvs12 followed by T. virens MTCC 794 (89.33%) and T. koningii MTCC 796 (62.39%). Microscopic study confirmed biocontrol mechanism as mycoparasitism for Tvs12 and antibiosis for T. koningii MTCC 796. The sclerotial biogenesis of test pathogen was elevated during weak antagonism and diminished in interactions with strong antagonists. The inhibition coefficient of S. rolfsii was significantly negatively correlated with sclerotia formation and lipid peroxidation during the antagonism. Trichoderma strains were screened for fungicides (carbendazim and tebuconazole, thiram and mancozeb) and abiotic stress (drought and salt) tolerance. Results indicated that T. koningii MTCC 796 efficiently grew better than the other strains with maximum radial growth under adverse conditions. The genetic variability among the Trichoderma was determined using 34 gene specific markers which amplified 146 alleles. The SSR similarities explained substantial diversity (15 to 87%) across Trichoderma strains and pathogen S. rolfsii. Principal coordinates analysis (PCA) were comparable to the cluster analysis and first three most informative PC components explained 64.45% of the total variation. In PCA, potent antagonists appear to be distinct from other strains. Five SSR markers T1F/T1R (311) , TvCTT56f/TvCTT56r (387) , TvGAT18f/TvGAT18r (364) , TvCA39f/TvCA39r (196) and TvAG29f/TvAG29r (418) found to be unique to distinguish best antagonist strain Tvs12. However, MTCC 796 was examined most stress tolerant strain with better inhibition coefficient which might be useful to control the disease under adverse conditions or as a part of integrated pest management., (Copyright © 2017. Published by Elsevier B.V.)

The study was performed to examine 11 isolates of Trichoderma for their bio-control potentials against Sclerotium rolfsii Sacc. causing stem rot in groundnut. The antagonists Trichoderma were subjected to sequence related amplified polymorphism (SRAP) based molecular diversity analysis and compared with their hardness to S. rolfsii with respect to secretary antifungal and antioxidant profile. T. virens NBAII Tvs 12 evident highest (87.91 %) growth inhibition of test pathogen followed by T. koningii MTCC 796 (67.03 %) at 7 days after inoculation (DAI). Microscopic study confirmed biocontrol mechanism as mycoparasitism for Tvs 12 and antibiosis for MTCC 796. The growth inhibition of test pathogen was significantly negatively correlated with sclerotia formation and lipid peroxidation during antagonism due to release of secretary bioactive antioxidants by antagonists to terminate oxidative burst generated by S. rolfsii and causing inhibition of sclerotium formation. The GC-MS profile identified antifungal and antioxidant constituents hexadecane, 1,2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester, 1-hexadecanesulfonyl chloride, and octadecane in potent antagonists Tvs 12; and nonacosane and octadecane in MTCC 796 along with two novel compounds 1-pentadecene and 1-heneicosyl formate for biocontrol activity. Molecular diversity of Trichoderma isolates associated with antagonistic activity was assessed by SRAP markers. The 115 primer combinations generate total 1328 amplified products of which, 1095 are shared polymorphic and 199 are unique polymorphic. The 15 SRAP combinations produced 18 bands to diagnose best antagonist Tvs 12 and 13 SRAP combinations generated 19 unique bands for identification of MTCC 796. The mycoparasitic antagonist Tvs 12 would be the best antagonist and released unique antifungal and antioxidant constituents to combat pathogen infection. The SRAP based genetic diversity indicates Tvs12 strain clustered with T. viride NBAII Tv23 and shared only 52 % similarity with other isolates of Trichoderma. The SRAP similarities explained substantial diversity (19-68 %) across Trichoderma isolates.

Microsatellites provide an ideal molecular markers system to screen, characterize and evaluate genetic diversity of several fungal species. Currently, there is very limited information on the genetic diversity of antagonistic Trichoderma species as determined using a range of molecular markers. In this study, expressed and whole genome sequences available in public database were used to investigate the occurrence, relative abundance and relative density of SSRs in five different antagonistic Trichoderma species: Trichoderma atroviride, T. harzianum, T. reesei, T. virens and T. asperellum. Fifteen SSRs loci were used to evaluate genetic diversity of twenty isolates of Trichoderma spp. from different geographical regions of India. Results indicated that relative abundance and relative density of SSRs were higher in T. asperellum followed by T. reesei and T. atroviride. Tri-nucleotide repeats (80.2%) were invariably the most abundant in all species. The abundance and relative density of SSRs were not influenced by the genome sizes and GC content. Out of eighteen primer sets, only 15 primer pairs showed successful amplification in all the test species. A total of 24 alleles were detected and five loci were highly informative with polymorphism information content values greater than 0.40, these markers provide useful information on genetic diversity and population genetic structure, which, in turn, can exploit for establishing conservation strategy for antagonistic Trichoderma isolates.

Background: Trichoderma is one of the most common fungi in soil. However, little information is available concerning the diversity of Trichoderma in soil with no previous history of cultivation. This study was conducted to investigate the most common species and the level of genetic relatedness of Trichoderma species from uncultivated soil in relation to cultivated soil and potting media., Results: A total of 24, 15 and 13 Trichoderma isolates were recovered from 84 potting media samples, 45 cultivated soil samples and 65 uncultivated soil samples, respectively. Analysis based on the internal transcribed spacer region of the ribosomal RNA (rRNA) and the translation elongation factor gene (EF1) indicated the presence of 9 Trichoderma species: T. harzianum (16 isolates), T. asperellum (13), T. citrinoviride (9), T. orientalis (3), T. ghanense (3), T. hamatum (3), T. longibrachiatum (2), T. atroviride (2), and T. viride (1). All species were found to occur in potting media samples, while five Trichoderma species were recovered from the cultivated soils and four from the uncultivated soils. AFLP analysis of the 52 Trichoderma isolates produced 52 genotypes and 993 polymorphic loci. Low to moderate levels of genetic diversity were found within populations of Trichoderma species (H = 0.0780 to 0.2208). Analysis of Molecular Variance indicated the presence of very low levels of genetic differentiation (Fst = 0.0002 to 0.0139) among populations of the same Trichoderma species obtained from the potting media, cultivated soil and uncultivated soil., Conclusion: The study provides evidence for occurrence of Trichoderma isolates in soil with no previous history of cultivation. The lack of genetic differentiation among Trichoderma populations from potting media, cultivated soil and uncultivated soil suggests that some factors could have been responsible for moving Trichoderma propagules among the three substrates. The study reports for the first time the presence of 4 Trichoderma species in Oman: T. asperellum, T. ghanense, T. longibrachiatum and T. orientalis.

Molecular markers that enable monitoring of fungi in their natural environment or assist in the identification of specific strains would facilitate Trichoderma utilization, particularly as an agricultural biocontrol agent (BCA). In this study, sequence analysis of internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of the ribosomal RNA (rRNA) gene cluster, a fragment of the translation elongation factor 1-alpha (tef1) gene, and random amplified polymorphic DNA (RAPD) markers were applied to determine the genetic diversity of Trichoderma atroviride strains collected in Poland, and also in order to identify loci and PCR-based molecular markers useful in genetic variation assessment of that fungus. Although tef1 and RAPD analysis showed limited genetic diversity among T. atroviride strains collected in Poland, it was possible to distinguish major groups that clustered most of the analyzed strains. Polymorphic RAPD amplicons were cloned and sequenced, yielding sequences representing 13 T. atroviride loci. Based on these sequences, a set of PCR-based markers specific to T. atroviride was developed and examined. Three cleaved amplified polymorphic sequence (CAPS) markers could assist in distinguishing T. atroviride strains. The genomic regions identified may be useful for further exploration and development of more precise markers suitable for T. atroviride identification and monitoring, especially in environmental samples.

Towards assessing the genetic diversity and occurrence of Trichoderma species from the Indian region of Indo-Burma Biodiversity hotspot, a total of 193 Trichoderma strains were isolated from cultivated soils of nine different districts of Manipur comprising 4 different agroclimatic zones. The isolates were grouped based on the morphological characteristics. ITS-RFLP of the rDNA region using three restriction digestion enzymes: Mob1, Taq1, and Hinf1, showed interspecific variations among 65 isolates of Trichoderma. Based on ITS sequence data, a total of 22 different types of representative Trichoderma species were reported and phylogenetic analysis showed 4 well-separated main clades in which T. harzianum was found to be the most prevalent spp. among all the Trichoderma spp. Combined molecular and phenotypic data leads to the development of a taxonomy of all the 22 different Trichoderma spp., which was reported for the first time from this unique region. All these species were found to produce different extrolites and enzymes responsible for the biocontrol activities against the harmful fungal phytopathogens that hamper in food production. This potential indigenous Trichoderma spp. can be targeted for the development of suitable bioformulation against soil and seedborne pathogens in sustainable agricultural practice.

The present study includes the molecular characteristics of Trichoderma pleurotum and Trichoderma pleuroticola isolates collected from green moulded cereal straw substrates at 47 oyster mushroom farms in Poland. The screening of the 80 Trichoderma isolates was performed by morphological observation and by using the multiplex PCR assay. This approach enabled specific detection of 47 strains of T. pleurotum and 2 strains of T. pleuroticola. Initial identifications were confirmed by sequencing the fragment of internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of the rRNA gene cluster and the fragment including the fourth and fifth introns and the last long exon of the translation-elongation factor 1-alpha (tef1) gene. ITS and tef1 sequence information was also used to establish the intra- and interspecies relationship of T. pleurotum and T. pleuroticola originating from the oyster mushroom farms in Poland and from other countries. Comparative analysis of the ITS sequences showed that all T. pleurotum isolates from Poland represent one haplotype, identical to that of T. pleurotum strains from Hungary and Romania. Sequence analysis of the tef1 locus revealed two haplotypes ("T" and "N") of Polish T. pleurotum isolates. The "T" type isolates of T. pleurotum were identical to those of strains from Hungary and Romania. The "N" type isolates possessed a unique tef1 allele. Detailed analysis of the ITS and tef1 sequences of two T. pleuroticola isolates showed their identicalness to Italian strain C.P.K. 1540.

In the present study, we reinvestigate the diversity of Trichoderma in Poland utilizing a combination of morphological and molecular/phylogenetic methods. A total of 170 isolates were collected from six different substrata at 49 sites in Poland. These were divided among 14 taxa as follows: 110 of 170 Trichoderma isolates were identified to the species level by the analysis of their ITS1, ITS2 rDNA sequences as: T. harzianum (43 isolates), T. aggressivum (35), T. citrinoviride (11), T. hamatum (9), T. virens (6), T. longibrachiatum (4), T. polysporum (1), and T. tomentosum (1); 60 isolates belonging to the Viride clade were identified based on a fragment of the translation-elongation factor 1-alpha (tef1) gene as: T. atroviride (20 isolates), T. gamsii (2), T. koningii (17), T. viridescens (13), T. viride (7), and T. koningiopsis (1). Identifications were made using the BLAST interface in TrichOKEY and TrichoBLAST ( http://www.isth.info ). The most diverse substrata were soil (nine species per 22 isolates) and decaying wood (nine species per 75 isolates). The most abundant species (25%) isolated from all substrata was T. harzianum.

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).

Towards assessing the biodiversity and biogeography of Trichoderma, we have analyzed the occurrence of Trichoderma species in soil and litter from four areas in China: North (Hebei province), South-East (Zhejiang province), West (Himalayan, Tibet) and South-West (Yunnan province). One hundred and thirty five isolates were grouped according to tentative morphological identification. A representative 64 isolates were verified at the species level by the oligonucleotide barcode program TrichO Key v.1.0 and the custom BLAST server TrichoBLAST, using sequences of the ITS1 and 2 region of the rRNA cluster and from the longest intron of the tef1 (translation elongation factor 1-alpha) gene. Eleven known species (Trichoderma asperellum, T. koningii, T. atroviride, T. viride, T. velutinum, T. cerinum, T. virens, T. harzianum, T. sinensis, T. citrinoviride, T. longibrachiatum) and two putative new species (T. sp. C1, and T. sp. C2), distinguished from known species both by morphological characters and phylogenetic analysis, were identified. A significant difference in the occurrence of these species was found between the North (Hebei) and South-West (Yunnan) areas, which correlates with previously reported species distributions in Siberia and South-East Asia, respectively. As in previous studies, T. harzianum accounted for almost half of the biodiversity; although, in this study, it was exclusively found in the North, and was predominantly represented by an ITS1 and 2 haplotype, which has so far been rarely found elsewhere. This study therefore reveals a North-South gradient in species distribution in East Asia, and identifies Northern China as a potential center of origin of a unique haplotype of T. harzianum.

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.

We investigated the occurrence and genetic diversity of Trichoderma in the river Danube national park, a primeval, riparian forest area located south-east of Vienna (Austria) which represents one of the last cases of an original European river-floodplain landscape. Forty-six strains were isolated and identified at the species level by analysis of morphological characters, by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS 1 and 2) of the rDNA cluster and--in some cases--a fragment of the translation elongation factor 1alpha (tef1) gene, and RAPD-analysis. Twenty-one strains were positively identified as T. harzianum, thirteen as T. rossicum, four as T. cerinum, two as T. hamatum, and one each as T. atroviride and T. koningii: four strains yielded two different ITS1 and 2 as well as tef1 sequence types, which were not alignable with any known species. Our studies show that they represent two new taxa of Trichoderma.