[Show abstract][Hide abstract] ABSTRACT: The generation of a wide ESTs library and database from Trichoderma harzianum CECT 2413 was the base for identifying the gene ThPTR2, coding for a PTR family di/tri-peptide transporter. The deduced protein sequence of the ThPTR2 gene showed the conserved motifs and also the 12 transmembrane domains typical of the PTR transporters. The highest level of ThPTR2 expression was found when the fungus was grown in chitin as sole carbon source. We also found that ThPTR2 expression was increased when Trichoderma interacted directly in solid medium with the plant-pathogenic fungus Botrytis cinerea, showing that ThPTR2 is involved in the mycoparasitic process. Additionally, its expression was triggered by nitrogen starvation and a higher level of expression was also found when Trichoderma was grown in secondary nitrogen sources like allantoin, yeast extract, and urea. However, no difference was found when Trichoderma was grown in presence or absence of glucose as carbon source. Strain T34-15, a transformant that overexpressed the ThPTR2 gene, showed about a 2-fold increase in the uptake of the dipeptide Leu-Leu. Additionally, two transformants from the strain Trichoderma longibrachiatum T52 that overexpressed ThPTR2 were also studied, confirming the role of this gene in peptide transport. Other homologous genes to ThPTR2 were identified in other Trichoderma strains. ThPTR2 is the first experimentally confirmed PTR family transporter gene from filamentous fungi.
Fungal Genetics and Biology 05/2006; 43(4):234-46. · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Different tissues of potato, tobacco, and bean plants were screened for anti-fungal protease inhibitor (PI) activity, also following fungal pathogen inoculation or mechanical wounding. A potato (Solanum tuberosum var. Desireé) sprout protein extract showed a strong inhibitory activity against chymotrypsin and Botrytis cinerea fungal proteases, but also on spore germination, hyphal elongation, and development of necrotic lesions. An active mixture of different proteins was affinity column purified and sequenced. Two new anti-fungal genes, PKI1 and PPI3B2, coding, respectively, for a Kunitz-type inhibitor and a Proteinase Inhibitor 1 capable of reducing fungal lesion development, were cloned and partially characterized. Direct effect on leaf necrosis formation was found to be dependent on the anti-chymotrypsin activity of both selected inhibitors. The PKI1 transcript was found to accumulate in untreated sprout tissues, although homologues of this gene seemed to accumulate following Bemisia tabaci attack. In the case of PPI3B2, we provide preliminary evidence that a member of the Proteinase Inhibitor 1 family is active against not only herbivorous insects but also phytopathogenic fungi and foliar lesions caused by them.
Physiological and Molecular Plant Pathology 04/2006; · 1.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trichoderma species are commonly used as biocontrol agents of different plant-pathogenic fungi. Terpene compounds are involved in the biocontrol process due to their antifungal properties (e.g., ergokonins and viridins) but additionally their structural function in the cell membranes (ergosterol) is essential. We report here the characterization of the T. harzianum erg1 gene, encoding a squalene epoxidase, a key enzyme in the biosynthesis of triterpene derivatives such as ergosterol. In T. harzianum the partial silencing of the erg1 gene gave rise to transformants with a higher level of sensitivity to terbinafine, an antifungal compound that acts specifically over the squalene epoxidase activity. In addition, these silenced transformants produced lower levels of ergosterol than the wild type strain. Finally, the silencing of the erg1 gene resulted in an increase in the expression level of the erg7 gene that encodes the oxidosqualene lanosterol-cyclase, another enzyme of the terpene biosynthesis pathway.
Fungal Genetics and Biology 04/2006; 43(3):164-78. · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The entomopathogenic anamorphic fungus Metarhizum anisopliae is currently used as a biocontrol agent (BCA) of insects. In the present work, we analyzed the sequence data obtained from group I introns in the large subunit (LSU) of rDNA genes with a view to determining the genetic diversity present in an autochthonous collection of twenty-six M. anisopliae isolates selected as BCAs.
DNA fragments corresponding to the 3'-end of the nuclear LSU rDNA genes of 26 M. anisopliae isolates were amplified by PCR. The amplicon sizes ranged from 0.8 to 3.4-kb. Four intron insertion sites, according to Escherichia coli J01695 numbering, were detected--Ec1921, Ec2066, Ec2449 and Ec2563--after sequencing and analysis of the PCR products. The presence/absence of introns allowed the 26 isolates to be distributed into seven genotypes. Nine of the isolates tested showed no introns, 4 had only one, 3 two, and 10 displayed three introns. The most frequent insertion sites were Ec1921 and Ec2449. Of the 26 isolates, 11 showed insertions at Ec2563 and a 1754-bp sequence was observed in ten of them. The most-parsimonious (MP) tree obtained from parsimony analysis of the introns revealed a main set containing four-groups that corresponded to the four insertion sites.
Four insertion sites of group I introns in the LSU rDNA genes allowed the establishment of seven genotypes among the twenty-six biocontrol isolates of M. anisopliae. Intron insertions at the Ec2563 site were observed for first time in this species.
[Show abstract][Hide abstract] ABSTRACT: The characterization of 11- and 18-residue peptaibols (peptides synthesized by peptide synthetases) at Trichoderma harzianum CECT 2413 (a filamentous fungus) was performed. Using a heterologous probe from tex1, the only peptaibol synthetase cloned and characterized so far in Trichoderma species, was cloned; a region that comprised 11676 bp of a second peptide synthetase gene detected in these strain (called salps2) and sequenced. The deduced sequence of Salps2 (3891 amino acids) contained three complete and a fourth incomplete module of a peptide synthetase, in which the typical adenylation, thiolation and condensation domains were found, but also an additional dehydrogenase/reductase domain in the C-terminus of the last module. Based on sequence similarity and analysis of its modular structure, it is proposed that Salps2 is a peptaibol synthetase. Additionally, analysis of =4.4-kb sequence downstream of salps2 was done and the signature sequences of Salps2 were identified and compared with those of available sequences of the other Trichoderma peptaibol synthetases.
[Show abstract][Hide abstract] ABSTRACT: The filamentous fungus Trichoderma harzianum is used as biological control agent of several plant-pathogenic fungi. In order to study the genome of this fungus, a functional genomics project called "TrichoEST" was developed to give insights into genes involved in biological control activities using an approach based on the generation of expressed sequence tags (ESTs).
Eight different cDNA libraries from T. harzianum strain CECT 2413 were constructed. Different growth conditions involving mainly different nutrient conditions and/or stresses were used. We here present the analysis of the 8,710 ESTs generated. A total of 3,478 unique sequences were identified of which 81.4% had sequence similarity with GenBank entries, using the BLASTX algorithm. Using the Gene Ontology hierarchy, we performed the annotation of 51.1% of the unique sequences and compared its distribution among the gene libraries. Additionally, the InterProScan algorithm was used in order to further characterize the sequences. The identification of the putatively secreted proteins was also carried out. Later, based on the EST abundance, we examined the highly expressed genes and a hydrophobin was identified as the gene expressed at the highest level. We compared our collection of ESTs with the previous collections obtained from Trichoderma species and we also compared our sequence set with different complete eukaryotic genomes from several animals, plants and fungi. Accordingly, the presence of similar sequences in different kingdoms was also studied.
This EST collection and its annotation provide a significant resource for basic and applied research on T. harzianum, a fungus with a high biotechnological interest.
[Show abstract][Hide abstract] ABSTRACT: Methanol extracts from 24 Trichoderma isolates, selected as biocontrol agents and representating different species and genotypes from three of the four taxonomic sections of this genus (T. sect. Trichoderma, T. sect. Pachybasium and T. sect. Longibrachiatum) were screened for antibacterial, anti-yeast and antifungal activities against a panel of seven bacteria, seven yeasts and six filamentous fungi previously used in similar studies. Two different growth media were tested (potato dextrose broth and CYS80), and all isolates included in the antimicrobial tests showed at least one inhibitory activity against one of the target microorganisms in one of the two culture media. No statistically significant differences were detected in the number of active strains between the two culture media, but the highest number of inhibitory strains against bacteria and fungi were found in strains from Trichoderma sect. Pachybasium, whereas strains from T. sect. Longibrachiatum showed the highest anti-yeast values. In all cases, a correlation was found between the strains that were active against yeasts and fungi. However, some degree of variability was detected for strains within the same taxonomic section. In general terms, strains from T. asperellum (mainly in CYS80 medium), and T. longibrachiatum gave the best non-enzymatic antimicrobial profiles.
Mycological Research 01/2006; 109(Pt 12):1397-406. · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A Gram-positive, aerobic, long-rod-shaped, non-spore-forming bacterium (strain PPLB(T)) was isolated from soil mixed with Iberian pig hair. This actinomycete showed keratinase activity in vitro when chicken feathers were added to the culture medium. Strain PPLB(T) was oxidase-negative and catalase-positive and produced lipase and esterase lipase. This actinomycete grew at 40 degrees C on nutrient agar and in the same medium containing 5 % (w/v) NaCl. Growth was observed with many different carbohydrates as the sole carbon source. On the basis of 16S rRNA gene sequence similarity, strain PPLB(T) was shown to belong to the genus Terrabacter of the family Intrasporangiaceae. Strain PPLB(T) showed 98.8 % 16S rRNA gene sequence similarity to Terrabacter tumescens. Chemotaxonomic data, such as the main ubiquinone (MK-8), the main polar lipids (phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylinositol) and the main fatty acids (i-C(15 : 0), ai-C(15 : 0), i-C(16 : 0) and ai-C(17 : 0)) supported the affiliation of strain PPLB(T) to the genus Terrabacter. The G+C content of the DNA was 71 mol%. The results of DNA-DNA hybridization (36.6 % relatedness between Terrabacter tumescens and strain PPLB(T)) and physiological and biochemical tests suggested that strain PPLB(T) belongs to a novel species of the genus Terrabacter, for which the name Terrabacter terrae sp. nov. is proposed. The type strain is PPLB(T) (=CECT 3379T=LMG 22921T).
International Journal of Systematic and Evolutionary Microbiology 12/2005; 55(Pt 6):2491-5. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trichoderma mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. We have analysed the extracellular proteome secreted by T. harzianum CECT 2413 in the presence of different fungal cell walls. Significant differences were detected in 2DE maps, depending on the use of specific cell walls or chitin. A combination of MALDI-TOF and liquid chromatography mass spectrometry allowed the identification of a novel aspartic protease (P6281: MW 33 and pI 4.3) highly induced by fungal cell walls. A broad EST library from T. harzianum CECT 2413 was used to obtain the full-length sequence. The protein showed 44% identity with the polyporopepsin (EC 220.127.116.11) from the basidiomycete Irpex lacteus. Lower identity percentages were found with other pepsin-like proteases from filamentous fungi (<31%) and animals (<29%). Northern blot and promoter sequence analyses support the implication of the protease P6281 in mycoparasitism.
Fungal Genetics and Biology 12/2005; 42(11):924-34. · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many currently available antifungal and antibacterial agents have undesirable toxic effects, and a wide spread use of these drugs has lead to rapid development of drug resistant strains which are the leading cause for treatment failure in both clinical and agricultural applications. The present article provides a synopsis of recent progress in investigations of new classes of antifungal compounds: disubstituted aliphatic and aromatic thioureas, triazole and thiazine compounds which act as ligands for transition metals. Antifungal effects of these compounds and selected metallic complexes versus representative plant pathogenic fungi are reviewed.
Journal of Inorganic Biochemistry 09/2005; 99(8):1558-72. · 3.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new component of the beta-1,6-glucanase (EC 18.104.22.168) multienzymatic complex secreted by Trichoderma harzianum has been identified and fully characterized. The protein, namely BGN16.3, is the third isozyme displaying endo-beta-1,6-glucanase activity described up to now in T. harzianum CECT 2413. BGN16.3 is an acidic beta-1,6-glucanase that is specifically induced by the presence of fungal cell walls in T. harzianum growth media. The protein was purified to electrophoretical homogenity using its affinity to beta-1,6-glucan as first purification step, followed by chomatofocusing and gel filtration. BGN16.3 has a molecular mass of 46 kDa in SDS/PAGE and a pI of 4.5. The enzyme only showed activity against substrates with beta-1,6-glycosidic linkages, and it has an endohydrolytic mode of action as shown by HPLC analysis of the products of pustulan hydrolysis. The expression profile analysis of BGN16.3 showed a carbon source control of the accumulation of the enzyme, which is fast and strongly induced by fungal cell walls, a condition often regarded as mycoparasitic simulation. The likely involvement beta-1,6-glucanases in this process is discussed.
[Show abstract][Hide abstract] ABSTRACT: Some of the secondary metabolites produced by Trichoderma, such as the peptaibols and other antibiotics, have a peptide structure and in their biosynthesis are involved proteins belonging to the Non-Ribosomal Peptide Synthetase family. In the present work, a PCR-mediated strategy was used to clone a region corresponding to an adenylation domain of a peptide synthetase (PS) gene from 10 different strains of Trichoderma. In addition, and using the fragment isolated by PCR from T. harzianum CECT 2413 as a probe, a fragment of 19.0 kb corresponding to a PS-encoding gene named salps1, including a 1.5 kb fragment of the promoter, was cloned and sequenced. The cloned region of salps1 contains four complete, and a fifth incomplete, modules, in which are found the adenylation, thiolation and condensation domains, but also an additional epimerization domain at the C-terminal end of the first module. The analysis of the Salps1 protein sequence, taking into consideration published data, suggests that it is neither a peptaibol synthetase nor a protein involved in siderophore biosynthesis. The presence of two breaks in the open reading frame and the expression of this gene under nitrogen starvation conditions suggest that salps1 could be a pseudogene.
[Show abstract][Hide abstract] ABSTRACT: Trichoderma species have been investigated as biological control agents for over 70 years owing to their ability to antagonize plant pathogenic fungi. Mycoparasitism, one of the main mechanisms involved in the antagonistic activity of Trichoderma strains, depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. The antifungal activity of an alpha-1,3-glucanase (EC 22.214.171.124, enzymes able to degrade alpha-1,3-glucans and also named mutanases) has been described in T. harzianum and its role in mycoparasitic processes has been suggested. In this study, we report on the purification, characterization and cloning of an exo-alpha-1,3-glucanase, namely AGN13.2, from the antagonistic fungus T. asperellum T32. Expression at the transcription level in confrontation assays against the strawberry pathogen Botrytis cinerea strongly supports the role of AGN13.2 during the antagonistic action of T. asperellum.
[Show abstract][Hide abstract] ABSTRACT: Strain identification in situ is an important factor in the monitoring of microorganisms used in the field. In this study, we demonstrated the use of sequence-characterized amplified region (SCAR) markers to detect genomic DNA from Trichoderma harzianum 2413 from soil. Two primers (SCAR A1/SCAR A1c) were tested against DNA of 27 isolates of Trichoderma spp. and amplified a 990-bp fragment from T. atroviride 11 and a 1.5-kb fragment from T. harzianum 2413, using an annealing temperature of 68 degrees C. These fragments showed no significant homology to any sequence deposited in the databases. The primer pair, BR1 and BR2, was designed to the 1.5-kb fragment amplified from T. harzianum 2413, generating a SCAR marker. To test the specificity of these primers, experiments were conducted using the DNA from 27 Trichoderma spp. strains and 22 field soil samples obtained from four different countries. PCR results showed that BR1 and BR2 amplified an 837-bp fragment unique to T. harzianum 2413. Assays in which total DNA was extracted from sterile and nonsterile soil samples, inoculated with spore or mycelium combinations of Trichoderma spp. strains, indicated that the BR1 and BR2 primers could specifically detect T. harzianum 2413 in a pool of mixed DNA. No other soil-microorganisms containing these sequences were amplified using these primers. To test whether the 837-bp SCAR marker of T. harzianum 2413 could be used in real-time PCR experiments, new primers (Q2413f and Q2413r) conjugated with a TaqMan fluorogenic probe were designed. Real-time PCR assays were applied using DNA from sterile and nonsterile soil samples inoculated with a known quantity of spores of Trichoderma spp. strains.
[Show abstract][Hide abstract] ABSTRACT: Trichoderma is known for being the most frequently used biocontrol agent in agriculture. A fundamental part of the Trichoderma antifungal system relies on a series of genes coding for a variety of extracellular lytic enzymes. Characterization of the polymorphism between five putative isoenzymatic activities [beta-1,3-glucanase (EC 126.96.36.199, EC 188.8.131.52), beta-1,6-glucanase (EC 184.108.40.206), cellulase (EC 220.127.116.11; EC 18.104.22.168, EC 22.214.171.124), chitinase (EC 126.96.36.199, EC 188.8.131.52), protease (EC 3.4.11; EC 3.4.13-19; EC 3.4.21-24, EC 3.4.99)] was carried out using 18 strains from three sections of Trichoderma. Of these, seven strains were from T. sect. Pachybasium, nine from T. sect. Trichoderma and two from T. sect. Longibrachiatum. Thirty-seven different alleles in total were identified: 13 for beta-1,3-glucanase, four for beta-1,6-glucanase, three for cellulase, eight for chitinase and nine for protease activity. A dendrogram (constructed by the unweighted pair group method with arithmetic averages) based on isoenzymatic data separated the 18 strains into three main enzymatic groups: T. harzianum, T. atroviride/T. viride/T. koningii and T. asperellum/T. hamatum/T. longibrachiatum. Isoenzymatic groupings obtained from biocontrol strains are discussed in relation to their phylogenetic location, based on their sequence of internal transcribed spacer 1 in ribosomal DNA and their antifungal activities.
Current Genetics 12/2004; 46(5):277-86. · 1.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetic variability within 69 biocontrol isolates of Trichoderma, obtained from different geographical locations and culture collections and selected as biocontrol agents, was studied. Sequence data, obtained from the ITS1 region of rDNA and a fragment of the translation elongation factor 1 (tef1) gene, were used in a phylogenetic analysis. Phylograms showing similar topologies were generated using alignments containing the ITS1 region or a portion of the tef1 gene. 21 distinct ITS1 sequence types and 17 distinct tef1 sequence types were identified among the 69 isolates. More than 50% of the potential biocontrol strains were grouped within Trichoderma sect. Pachybasium; of these, 81% were grouped within the cluster that included the ex-type strains of T. harzianum and T. inhamatum, and 16% were grouped with T. virens. Within T. sect. Trichoderma, which included 36% of the 69 strains, 56% were grouped with T. asperellum, and 24% with T. viride, T. atroviride or T. koningii. Only 10% of the strains studied were located in T. sect. Longibrachiatum.
Mycological Research 09/2004; 108(Pt 8):897-906. · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The thiourea derivatives of N-butylmethylamine (3-benzoyl-1-butyl-1-methyl-thiourea) (1), N-ethylisopropylamine (3-benzoyl-1-ethyl-1-isopropyl-thiourea) (2) and the corresponding complexes of 1 and 2 with Ni(II), Co(III) and Pt(II) have been synthesized. The compounds obtained were characterized by elemental analysis, spectroscopic methods (FT-IR, UV-Vis and NMR) and mass spectrometry. Compound 1, crystallized in the triclinic space group. The antifungal activities of compounds 1 and 2 and their corresponding complexes against the fungus Penicillium digitatum and against the yeast Saccharomyces cerevisiae were investigated. In general, fungal growth inhibition was higher with compound 1 and its complexes than with compound 2, except for the Co(III) complex of 2.
Journal of Inorganic Biochemistry 09/2004; 98(8):1307-14. · 3.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mycoparasitic Trichoderma strains secrete a complex set of hydrolytic enzymes under conditions related to antagonism. Several proteins with proteolytic activity were detected in culture filtrates from T. harzianum CECT 2413 grown in fungal cell walls or chitin and the protein responsible for the main activity (PRA1) was purified to homogeneity. The enzyme was monomeric, its estimated molecular mass was 28 kDa (SDS-PAGE), and its isoelectric point 4.7-4.9. The substrate specificity and inhibition profile of the enzyme correspond to a serine-protease with trypsin activity. Synthetic oligonucleotide primers based on N-terminal and internal sequences of the protein were designed to clone a full cDNA corresponding to PRA1. The protein sequence showed <43% identity to mammal trypsins and 47-57% to other fungal trypsin-like proteins described thus far. Northern analysis indicated that PRA1 is induced by conditions simulating antagonism, is subject to nitrogen and carbon derepression, and is affected by pH in the culture media. The number of hatched eggs of the root-knot nematode Meloidogyne incognita was significantly reduced after incubation with pure PRA1 preparations. This nematicidal effect was improved using fungal culture filtrates, suggesting that PRA1 has additive or synergistic effects with other proteins produced during the antagonistic activity of T. harzianum CECT 2413. A role for PRA1 in the protection of plants against pests and pathogens provided by T. harzianum CECT 2413 is proposed.
Applied Microbiology and Biotechnology 07/2004; 65(1):46-55. · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trichoderma strains are considered to be among the most useful fungi in industrial enzyme production, agriculture and bioremediation. Much less developed is the idea of looking at these micromycetes as model microorganisms to study and improve the understanding of important microbial interactions, for instance with plants and pests. In fact, the use of genomic approaches to study the complex and fascinating mechanisms that permit Trichoderma to produce large amount of heterologous proteins (this aspect will be reviewed in detail in another chapter of this book), control pathogens and effect plant metabolism and physiology is still in its infancy. Very little has been accomplished to date, even though the need and interest in sustaining both structural and functional genomic projects is widely recognized and has led to funding and start up of several new initiatives. This chapter presents the rationale used for investing in a wide genome effort on these fungi, based on diversity and potential utility of their biological characteristics, and discusses the problems related to the taxonomical discrimination of the various species and strains within this genus. It also describes the methods that have been used to date to obtain useful genomic data especially on T. reesei and a few other Trichoderma species. Finally, we describe the strategy and the preliminary results of a functional genomic project recently undertaken by an International Consortium comprised of academic institutions and small enterprises, which was purposely formed to follow this initiative. This program aims at exploring genetic biodiversity within the Trichoderma genus, providing basic knowledge on the biology of these complex microorganisms, and developing commercial applications in agriculture, food industry and medicine.