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[Presearch on preventing the medicinal plant diseases with Trichoderma harzianum preparation].

OBJECTIVE: To control the medicinal plant diseases with the preparation of Trichoderma harzianum. METHOD: Antagonistic action of the preparation to the pathogens of the medicinal plants in vitro, and controling effects of the preparation on these diseases in greenhouse and in the field were tested. RESULT: The test in vitro showed that Trichoderma harzianum, used as a biocontrol factor, had stronger antagonistic action to Fusarium equiseti, Sclerotinia sp. and Rhizoctonia solani which were the medicinal plant pathogens of Astragalus membranaceus, Glehnia littoralis and Panax quinquefolium respectively. Biological controling effects on sclerotium root rot of Glehnia littoralis were 83.6% and 72.5% respectively in greenhouse and in the field with the preparation of Trichoderma harzianum. And controling effects on root rot of Astragalus membranaceus and seedling damping-off of Panax quinquefolium were 80% and 60% respectively in the field. The dosage of the preparation used in the field was 10 g.m-2. CONCLUSION: The preparation of Trichoderma harzianum can be used as a substitute for such chemicals as Carbendazim. Using the preparation to control medicinal plant diseases provides a technical safeguard for the good agricultural practice of medicinal plants.

Apiaceae↗

[A study of the physicochemical and biological properties of mutanase from Trichoderma harzianum].

OBJECTIVE: To determine the physicochemical properties of the mutanase of Trichoderma harzianum isolated from China and to study the influence of mutanase on the adherence of oral Streptococci and the structure of oral biofilms. METHODS: Six fungal strains belonging to Trichoderma were tested for mutanase production in the same cultural condition, the strain producing the highest mutanase activity was studied further and the pH and temperature optimum of the enzyme was determined. The RT-PCR method was used to obtain the gene coding for mutanase and the product was cloned to pMD18-T simple vector for sequencing. Inhibition effects of mutanase on the adherence of Streptococcus sobrinus OMZ176, Streptococcus sobrinus 6715, Streptococcus mutans MT8148 were studied by adherence test. The optical sectioning of biofilms with or without mutanase supplementation were analyzed by confocal laser scanning microscopy (CLSM). RESULTS: The highest enzymatic activity was achieved by Trichoderma harzianum Th1, the maximum activity was at pH 5.5 and at 40 degrees C. The nucleotide sequence was 92% homology with that of a known gene coding a mutanase (GenBank accession No. AJ243799). The adherence of Streptococcus sobrinus OMZ176, Streptococcus sobrinus 6715, Streptococcus mutans MT8148 was significantly inhibited by mutanase. Compared with control, the biofilms with mutanase supplementation had lower height and sparser structure. CONCLUSIONS: The mutanase from Trichoderma harzianum Th1 can inhibit the adherence of oral Streptococci and had an influence on the structure of oral biofilms.

Bacterial Adhesion↗

Suppression of Fusarium wilt by combining green compost and Trichoderma hamatum.

Fusarium wilts, caused by the fungus Fusarium oxysporum, are important diseases of horticultural and agricultural crops and lead to significant yield losses. The pathogen infects the roots and colonizes the vascular tissue, leading to wilting and finally death of the plant. The objective of this study was to investigate the efficacy of amendment of green compost and a Trichoderma hamatum strain against Fusarium wilt of radish. The substrate effects and the effect of a Trichoderma strain were tested in a potting soil bioassay. The tested composts lowered the disease level and had a positive influence on the plant yield (fresh weight and dry weight). Nothwithstanding, only a small dosis effect of the amendment was observed. In the presence of the tested Trichoderma hamatum strain no significant lower disease level was observed. Furthermore, a minor effect on plant yield (fresh weight and dry weight) was observed compared to the amendment with only green compost. Maybe the pathogenic Fusarium oxysporum strain and the Trichoderma strain competed for nutrients, iron could be a limiting factor. One possible approach to improve biological control may be the application of combinations of biocontrol agents.

Antibiosis↗

Detection of peptaibols and their hydrolysis products in cultures of Trichoderma species.

Fifteen strains of Trichoderma belonging to 12 different species for potential use in biological control were examined for their ability to produce polypeptide antiobitics (peptaibols). Gas chromatography was used to detect the hydrolysis products of peptaibols after esterification with n-propanol and derivatisation with heptafluorobutyric anhydride. In particular, amino acids (alpha-aminoisobutyric acid, and isovalin) and amino alcohols (phenylalaninol, leucinol, and valinol) were used as markers for the detection of different peptaibols. alpha-Aminoisobutyric acid, an amino acid characterising all peptaibols, was detected in all cultures examined, indicating that the production of peptaibols is a common feature of the various Trichoderma species. Different amino acid/amino alcohol combinations suggested specific peptaibol production. On this basis, the peptaibol paracelsin was confirmed by HPTLC in 11 out of 15 Trichoderma strains cultures. Valinol was not found in any culture extract, indicating the lack of trichotoxins production. Culture extracts of the 6 tested Trichoderma strains were extremely toxic to Artemia salina larvae; for 5 of them this toxicity could be attributed to paracelsin (calculated LD50 = 2.2 microM).

Alamethicin↗

Successful treatment of a brain abscess due to Trichoderma longibrachiatum after surgical resection.

A case of brain abscess due to Trichoderma longibrachiatum in a leukemic patient with prolonged neutropenia is reported. Definitive cure was achieved after neurosurgical resection of the abscess and prolonged antifungal therapy. Trichoderma is a filamentous fungus species, which is only exceptionally pathogenic in humans. This genus and particularly the species Trichoderma longibrachiatum should be added to the growing list of fungi causing infection in immunocompromised patients.

Adolescent↗

Efficient production of laccases by Trametes sp. AH28-2 in cocultivation with a Trichoderma strain.

A biocontrol fungus isolated from rotting wood was identified as a Trichoderma strain (named as Trichoderma sp. ZH1) by internal transcribed spacer (ITS) sequences of rRNA genes. The laccase yield of Trametes sp. AH28-2 in cocultivation with Trichoderma sp. ZH1 reached 6,210 U l(-1), approximately identical to those induced by toxic aromatic inducers. Cocultures maintained 60-70 % of their highest laccase activity obtained at 5 days after inoculation of the biocontrol fungus, at least for 20 days. Furthermore, a novel laccase isozyme (LacC) was obtained through the fungal interactions. The molecular weight of LacC is about 64 kDa, and its isoelectric point is 6.6. The temperature and pH optimum for LacC to oxidize guaiacol are 55 degrees C and 5.0, respectively. LacC is stable both at 60 degrees C and pH 4.0-8.0. Furthermore, the K (m) values of LacC for various substrates were also determined. Our work demonstrates a safe strategy for the production of industrial laccases, instead of the traditional method of chemical induction.

DNA, Fungal↗

Biochemical characterization of a beta-1,3-glucanase from Trichoderma koningii induced by cell wall of Rhizoctonia solani.

Trichoderma species are readily isolated from Brazilian cerrado soil by conventional methods and some of them were characterized as Trichoderma koningii. The effect of carbon source on the production of beta-1,3-glucanases in the culture filtrates of a specific Trichoderma koningii strain (ALL 13) was investigated. Enzyme activity was detected in all carbon sources tested and only one band of beta-1,3-glucanase was detected in non-denaturing PAGE. This enzyme was purified by Sephacryl S-200 gel filtration and Phenyl Sepharose CL 4B chromatography. A typical procedure provided 105-fold purification with 13.4% yield. The molecular weight of the purified enzyme was 75 kDa as estimated by SDS-PAGE. The enzyme hydrolyzed laminarin in an endo-like fashion to form small oligosaccharides and glucose. The Km and Vmax values for beta-1,3-glucanase, using laminarin as substrate, were 0.148 mg.mL-1 and 0.159 U.min-1, respectively. The pH optimum for the enzyme was pH 4.6 and maximum activity was obtained at 50 degrees C. Hg2+ inhibited the purified enzyme.

Carbon↗

Fungal proteomics: mapping the mitochondrial proteins of a Trichoderma harzianum strain applied for biological control.

Mitochondria are essential for cellular functions across organisms. A proteomic approach was taken to separate and identify mitochondrial proteins from a strain of Trichoderma harzianum with well established biocontrol properties. We optimized a method for the preparation of a sample enriched with mitochondria by ensuring efficient cell lysis and including several washing steps to minimize cytoplasmic contamination. We separated hundreds of proteins, using two-dimensional gel electrophoresis and identified 31 protein spots from T. harzianum, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and liquid chromatrography with mass spectrometry. Over 50% of the identified proteins were known to localize in the mitochondria. Three protein spots were identified from T. harzianum and a further five protein spots from the genera Trichoderma and Hypocrea. The remaining protein spots were identified by cross-species identification from other filamentous fungi, including Neurospora crassa and Aspergillus spp, and yeasts, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. In total, we identified 25 protein spots from T. harzianum, representing proteins that have not been characterized in existing Trichoderma protein databases. To our knowledge, this is the first two-dimensional mitochondrial protein map of a filamentous fungus.

Chromatography, Liquid↗

Development of a strain-specific SCAR marker for the detection of Trichoderma atroviride 11, a biological control agent against soilborne fungal plant pathogens.

The genus Trichoderma includes biocontrol agents (BCAs) effective against soilborne plant pathogenic fungi. Several potentially useful strains for biological control are difficult to distinguish from other strains of Trichoderma found in the field. So, there is a need to find ways to monitor these strains when applied to natural pathosystems. We have used random amplified polymorphic DNA (RAPD) markers to estimate genetic variation among sixteen strains of the species T. asperellum, T. atroviride, T. harzianum, T. inhamatum and T. longibrachiatum previously selected as BCAs, and to obtain fingerprinting patterns. Analysis of these polymorphisms revealed four distinct groups, in agreement with previous studies. Some of the RAPD products generated were used to design specific primers. Diagnostic PCR performed using these primers specifically identify the strain T. atroviride 11, showing that DNA markers may be successfully used for identification purposes. This SCAR (sequence-characterised amplified region) marker can clearly distinguish strain 11 from other closely related Trichoderma strains.

Genetic Markers↗

The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source.

The gene pdi1 encoding protein disulphide isomerase was isolated from the filamentous fungus Trichoderma reesei by degenerate PCR based on a consensus PDI active-site sequence. It was shown that the Trichoderma pdi1 cDNA is able to complement a yeast mutant with a disrupted PDI1 gene. The putative T. reesei PD1I protein has a predicted 20-amino acid N-terminal signal sequence and the C-terminal fungal consensus ER retention signal HDEL. The mature protein shows strong conservation relative to other fungal protein disulphide isomerases. The T. reesei pdi1 promoter has two possible unfolded protein response (UPR) elements and it was shown by treatments with dithiothreitol and tunicamycin that the gene is under the control of the UPR pathway. Expression of a heterologous protein, an IgG antibody Fab fragment, in Trichoderma increases pdi1 expression, probably by inducing the UPR. The level of T. reesei pdi1 mRNA is also regulated by the carbon source, being lowest in glucose-containing media and highest on carbon sources that induce the genes encoding extracellular enzymes. The mechanism of this regulation was studied by examining pdi1 mRNA levels under conditions where the extracellular enzymes are induced by sophorose, as well as in the strain RutC-30, which is mutant for the glucose repressor gene cre1. The results suggest that neither sophorose induction nor glucose repression by the CREI protein affect the pdi1 promoter directly.

Amino Acid Sequence↗

Molecular characterization of a novel partitivirus harboring an additional third dsRNA segment from Trichoderma harzianum.

We report the complete genome sequence of a novel partitivirus identified from Trichoderma harzianum NFCF092 strain, designated Trichoderma harzianum partitivirus 4 (ThPV4). Unlike canonical members of the family Partitiviridae, which possess a bipartite genome consisting of two double-stranded RNA (dsRNA) segments encoding an RNA-dependent RNA polymerase (RdRP) and a capsid protein (CP), ThPV4 harbors a third dsRNA segment encoding a protein of unknown function. The complete genome consists of dsRNA1 (1,950 bp; encoding the RdRP), dsRNA2 (1,772 bp; encoding the CP), and dsRNA3 (1,629 bp; encoding a protein with unknown function). Sequence analysis shows that each segment possesses a single open reading frame (ORF). The deduced amino acid sequence of the RdRP shows the highest similarity (90.5% identity) to that of Trichoderma gamsii alphapartitivirus 1. Phylogenetic analyses based on the RdRP indicate that ThPV4 clusters within the genus Alphapartitivirus of the family Partitiviridae. To our knowledge. ThPV4 is the first member of the genus Alphapartitivirus identified from T. harzianum to possess an additional, conserved third dsRNA segment.

Phylogeny↗

Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum.

Protoplasts were isolated from Trichoderma harzianum strain PTh18 using lysing enzymes and self-fusion of T. harzianum protoplasts was carried out using polyethylene glycol in STC buffer. The fused protoplasts of T. harzianum were regenerated and 15 self-fusants were selected to study the chitinase production and biocontrol activity. High chitinase activity was measured in the culture filtrates of most of the self-fusants (87%) than the parent. Among the fusants, the strain SFTh8 produced maximum chitinase with a two-fold increase as compared to the parent strain. All the self-fusants exhibited increased antagonistic activity against Rhizoctonia solani than the parent. The crude chitinase preparation of SFTh8 lysed the mycelia of T. harzianum, Trichoderma viride and Trichoderma reesei and released the protoplasts in higher number than the crude chitinase preparation of parent strain PTh18.

Chitinases↗

Identification of novel Trichoderma hamatum genes expressed during mycoparasitism using subtractive hybridisation.

Subtractive hybridisation was used to target novel genes involved in the mycoparasitic interaction of the biocontrol agent Trichoderma hamatum with the phytopathogen Sclerotinia sclerotiorum. Nineteen novel T. hamatum genes were identified that showed increased expression during mycoparasitism compared to a T. hamatum control. Sequence analysis revealed some cDNA fragments had similarity to known fungal or bacterial genes whereas others had no similarity to any genes previously described. Only one of the novel genes has been characterised in another Trichoderma species, the Trichoderma reesei hex1gene. The proteins encoded by the novel genes included three monooxygenases, a metalloendopeptidase, a gluconate dehydrogenase, an endonuclease and a proton ATPase.

Adaptation, Physiological↗

Trichoderma biodiversity in China: evidence for a North to South distribution of species in East Asia.

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.

China↗

Trichoderma specialized metabolites in biocontrol: gene-metabolite links, ecological functions, and translational bottlenecks.

Trichoderma spp. produce a diverse repertoire of metabolites with specific activities that contribute to biocontrol through direct antagonism, ecological signalling, and modulation of plant responses. However, current knowledge remains uneven: many metabolites are chemically described, whereas fewer are supported by robust gene-metabolite associations, experimentally validated ecological functions, and realistic translational evidence. Progress in this field will depend less on expanding compound catalogues than on integrating mechanistic, ecological, and translational evidence. This review examines the specialized metabolism of Trichoderma with emphasis on biosynthetic gene clusters, regulatory networks, ecological roles, and biosafety constraints relevant to biocontrol. Major metabolite classes, including polyketides, terpenoids, peptaibols, siderophores, diketopiperazines, and volatile organic compounds, are discussed together with representative case studies for which genetic and functional evidence is available. We further propose a translational framework to distinguish metabolites with mainly descriptive support from those approaching application readiness, based on four criteria: gene-level validation, demonstrated ecological role, manageable biosafety profile, and feasible delivery/stability. This perspective helps explain why metabolite inventories continue to expand faster than field translation. Recent advances in genomics, transcriptomics, metabolomics, genome editing, and formulation science are reshaping how Trichoderma metabolites are prioritized for future development.

Biosafety↗

SCAR-based real time PCR to identify a biocontrol strain (T1) of Trichoderma atroviride and study its population dynamics in soils.

Strains of Trichoderma spp. are known for their antagonistic properties against plant pathogens, some are already on the market, others are under development. In order to launch a strain on the market its perfect identification at the species and strain levels is needed. The aim of this study is to (i) design a SCAR marker for specific identification of strain T1 of Trichoderma atroviride and (ii) monitor population dynamics of this strain in soil by real time PCR. A primer pair targeting a 141-bp fragment enabled specific detection of this strain without cross detection of autochthonous populations of Trichoderma in several field soils. In two soils, population dynamics assessed by real time PCR and the soil plate technique gave similar results. The molecular tools developed in this study satisfy the requirement for specific identification of the biocontrol strain and for detection and quantification of T. atroviride T1 population in complex environments.

DNA Fingerprinting↗

Identification of genes with a biocontrol function in Trichoderma harzianum mycelium using the expressed sequence tag approach.

Species of Trichoderma are commercially applied as biological control agents against plant fungal pathogens based on different mechanisms, such as the production of antifungal metabolites, competition for space and nutrients and mycoparasitism. But the integrated biocontrol mechanism of Trichoderma harzianum is not well explored at the genetic level. This study aimed to initiate the preliminary development of an expressed sequence tag (EST) database for T. harzianum and thereby gain potentially useful information on Trichoderma gene sequences in order to elucidate the integrated biocontrol mechanism. Partial sequencing of anonymous cDNA clones is a widely used technique for gene identification. A directional cDNA library has been constructed from mycelium of T. harzianum and 3298 clones have been randomly selected, subjected to single-pass sequencing from the 5' end of the vector, and identified by sequence similarity searches against gene sequences in international databases. Of the 3298 mycelium clones, 2174 exhibit similarity to known genes and 451 to known ESTs, while 673 represent novel gene sequences. Analysis of the identified clones indicated sequence similarity to a broad diversity of genes encoding proteins such as enzymes, structural proteins, and regulatory factors. A significant proportion of genes identified in the mycelium were involved in processes related to mycoparasitism and fungicidal metabolites, as would be expected in biocontrol fungus. These results present the successful application of EST analysis in T. harzianum and provide a preliminary indication of gene expression in mycelium.

Base Sequence↗

Acetyl xylan esterase II from Penicillium purpurogenum is similar to an esterase from Trichoderma reesei but lacks a cellulose binding domain.

Penicillium purpurogenum produces at least two acetyl xylan esterases (AXE I and II). The AXE II cDNA, genomic DNA and mature protein sequences were determined and show that the axe 2 gene contains two introns, that the primary translation product has a signal peptide of 27 residues, and that the mature protein has 207 residues. The sequence is similar to the catalytic domain of AXE I from Trichoderma reesei (67% residue identity) and putative active site residues are conserved, but the Penicillium enzyme lacks the linker and cellulose binding domain, thus explaining why it does not bind cellulose in contrast to the Trichoderma enzyme. These results point to a possible common ancestor gene for the active site domain, while the linker and the binding domain may have been added to the Trichoderma esterase by gene fusion.

Acetylesterase↗