Extracellular vesicles from the mycoparasitic fungus Trichoderma harzianum.

Trichoderma harzianum is a filamentous fungus that can act as a mycoparasite, saprophyte, or a plant symbiotic. It is widely used as a biological control agent against phytopathogenic fungi and can also be used for plant growth promotion and biofortification. Interaction between T. harzianum and phytopathogenic fungi involves mycoparasitism, competition, and antibiosis. Extracellular vesicles (EVs) have been described as presenting a central role in mechanisms of communication and interaction among fungus and their hosts. In this study, we characterized extracellular vesicles of T. harzianum produced during growth in the presence of glucose or S. sclerotiorum mycelia. A set of vesicular proteins was identified using proteomic approach, mainly presenting predicted signal peptides.

Trichoderma harzianum transcriptome in response to cadmium exposure.

Cadmium (Cd) is a heavy metal present in the environment mainly as a result of industrial contamination that can cause toxic effects to life. Some microorganisms, as Trichoderma harzianum, a fungus used in biocontrol, are able to survive in polluted environments and act as bioremediators. Aspects about the tolerance to the metal have been widely studied in other fungi although there are a few reports about the response of T. harzianum. In this study, we determined the effects of cadmium over growth of T. harzianum and used RNA-Seq to identify significant genes and processes regulated in the metal presence. Cadmium inhibited the fungus growth proportionally to its concentration although the fungus exhibited tolerance as it continued to grow, even in the highest concentrations used. A total of 3767 (1993 up and 1774 down) and 2986 (1606 up and 1380 down) differentially expressed genes were detected in the mycelium of T. harzianum cultivated in the presence of 1.0 mg mL-1 or 2.0 mg mL-1 of CdCl2, respectively, compared to the absence of the metal. Of these, 2562 were common to both treatments. Biological processes related to cellular homeostasis, transcription initiation, sulfur compound biosynthetic and metabolic processes, RNA processing, protein modification and vesicle-mediated transport were up-regulated. Carbohydrate metabolic processes were down-regulated. Pathway enrichment analysis indicated induction of glutathione and its precursor's metabolism. Interestingly, it also indicated an intense transcriptional induction, especially by up-regulation of spliceosome components. Carbohydrate metabolism was repressed, especially the mycoparasitism-related genes, suggesting that the mycoparasitic ability of T. harzianum could be affected during cadmium exposure. These results contribute to the advance of the current knowledge about the response of T. harzianum to cadmium exposure and provide significant targets for biotechnological improvement of this fungus as a bioremediator and a biocontrol agent.

Diversity and Pathogenicity of Rhizoctonia Species from the Brazilian Cerrado.

Eighty-one Rhizoctonia-like isolates were identified based on morphology and nuclei-staining methods from natural and agricultural soils of the Cerrado (Brazilian savanna). The nucleotide similarity analysis of ITS1-5.8S-ITS2 regions identified 14 different taxa, with 39.5% of isolates assigned to Waitea circinata (zeae, oryzae, and circinata varieties), while 37.0% belonged to Thanatephorus cucumeris anastomosis groups (AGs) AG1-IB, AG1-ID, AG1-IE, AG4-HGI, and AG4-HGIII. Ceratobasidium spp. AG-A, AG-F, AG-Fa, AG-P, and AG-R comprised 23.5%. Rhizoctonia zeae (19.8%), R. solani AG1-IE (18.6%), and binucleate Rhizoctonia AG-A (8.6%) were the most frequent anamorphic states found. Root rot severity caused by the different taxa varied from low to high on common beans, and tended to be low to average in maize. Twenty-two isolates were pathogenic to both hosts, suggesting difficulties in managing Rhizoctonia root rots with crop rotation. These results suggest that cropping history affects the geographical arrangement of AGs, with a prevalence of AG1 in the tropical zone from central to north Brazil while the AG4 group was most prevalent from central to subtropical south. W. circinata var. zeae was predominant in soils under maize production. To our knowledge, this is the first report on the occurrence of W. circinata var. circinata in Brazil.

Secretome analysis of the mycoparasitic fungus Trichoderma harzianum ALL 42 cultivated in different media supplemented with Fusarium solani cell wall or glucose.

Trichoderma harzianum is a fungus well known for its potential as a biocontrol agent against many fungal phytopathogens. The aim of this study was to characterize the proteins secreted by T. harzianum ALL42 when its spores were inoculated and incubated for 48 h in culture media supplemented with glucose (GLU) or with cell walls from Fusarium solani (FSCW), a phytopathogen that causes severe losses in common bean and soy crops in Brazil, as well as other crop diseases around the world. Trichoderma harzianum was able to grow in Trichoderma Liquid Enzyme Production medium (TLE) and Minimal medium (MM) supplemented with FSCW and in TLE+GLU, but was unable to grow in MM+GLU medium. Protein quantification showed that TLE+FSCW and MM+FSCW had 45- and 30- fold, respectively, higher protein concentration on supernatant when compared to TLE+GLU, and this difference was observable on 2D gel electrophoresis (2DE). A total of 94 out of 105 proteins excised from 2DE maps were identified. The only protein observed in all three conditions was epl1. In the media supplemented with FSCW, different hydrolases such as chitinases, ß-1,3-glucanases, glucoamylases, α-1,3-glucanases and proteases were identified, along with other proteins with no known functions in mycoparasitism, such as npp1 and cys. Trichoderma harzianum showed a complex and diverse arsenal of proteins that are secreted in response to the presence of FSCW, with novel proteins not previously described in mycoparasitic-related studies.

Application of lipase from Burkholderia cepacia in the degradation of agro-industrial effluent.

This study aimed to analyze the physical and chemical characteristics of Amano PS commercial lipase - Burkholderia cepacia and lipase produced by Burkholderia cepacia strain ATCC 25416, in addition to studying the hydrolysis of agro-industrial effluent collected in a fried potato industry. The optimum temperature for increasing lipase activity was 37 °C. The temperature increase caused a decrease in thermostability of lipase, and the commercial lipase was less stable, with values of 10.5, 4.6 and 4.9%, respectively, lower than those obtained by lipase from strain ATCC 25416, at temperatures of 40, 50 and 60 °C. The enzymatic activity was higher in alkaline conditions, achieving better results at pH 8.0. The pH was the variable that most influenced the hydrolysis of triacylglycerides of the agro-industrial effluent, followed by enzyme concentration, and volume of gum arabic used in the reaction medium. Thus, it can be observed that the enzymatic hydrolytic process of the studied effluent presents a premising contribution to reduction of environmental impacts of potato chip processing industries.

Identification of mycoparasitism-related genes against the phytopathogen Sclerotinia sclerotiorum through transcriptome and expression profile analysis in Trichoderma harzianum.

BACKGROUND: The species of T. harzianum are well known for their biocontrol activity against plant pathogens. However, few studies have been conducted to further our understanding of its role as a biological control agent against S. sclerotiorum, a pathogen involved in several crop diseases around the world. In this study, we have used RNA-seq and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum gene expression during growth on cell wall of S. sclerotiorum (SSCW) or glucose. RT-qPCR was also used to examine genes potentially involved in biocontrol, during confrontation between T. harzianum and S. sclerotiorum. RESULTS: Data obtained from six RNA-seq libraries were aligned onto the T. harzianum CBS 226.95 reference genome and compared after annotation using the Blast2GO suite. A total of 297 differentially expressed genes were found in mycelia grown for 12, 24 and 36 h under the two different conditions: supplemented with glucose or SSCW. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on SSCW or glucose. We identified various genes of biotechnological value encoding proteins with functions such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. To validate the expression profile, RT-qPCR was performed using 20 randomly chosen genes. RT-qPCR expression profiles were in complete agreement with the RNA-Seq data for 17 of the genes evaluated. The other three showed differences at one or two growth times. During the confrontation assay, some genes were up-regulated during and after contact, as shown in the presence of SSCW which is commonly used as a model to mimic this interaction. CONCLUSIONS: The present study is the first initiative to use RNA-seq for identification of differentially expressed genes in T. harzianum strain TR274, in response to the phytopathogenic fungus S. sclerotiorum. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against S.sclerotiorum. The RNA-seq data presented will facilitate improvement of the annotation of gene models in the draft T. harzianum genome and provide important information regarding the transcriptome during this interaction.

Mycoparasitism studies of Trichoderma harzianum against Sclerotinia sclerotiorum: evaluation of antagonism and expression of cell wall-degrading enzymes genes.

Trichoderma spp. are known for their biocontrol activity against several plant pathogens. A specific isolate of Trichoderma harzianum, 303/02, has the potential to inhibit the growth of Sclerotinia sclerotiorum, an important agent involved in several crop diseases. In this study, the interaction between T. harzianum 303/02 and mycelia, sclerotia and apothecia of S. sclerotiorum was studied by scanning electron microscopy. RT-qPCR was used to examine the expression of 11 genes potentially involved in biocontrol. T. harzianum 303/02 parasitizes S. sclerotiorum by forming branches that coil around the hyphae. The fungus multiplied abundantly at the sclerotia and apothecia surface, forming a dense mycelium that penetrated the inner surface of these structures. The levels of gene expression varied according to the type of structure with which T. harzianum was interacting. The data also showed the presence of synergistic action between the cell-wall degrading enzymes.

Cloning and characterization of a protein elicitor Sm1 gene from Trichoderma harzianum.

A small protein, cysteine-rich, designated SM1, produced by Trichoderma virens and Trichoderma atroviride, acts as elicitor for triggering plant defense reactions. We analyzed Sm1 gene expression of eight different strains of Trichoderma spp. grown on glucose, seeds or roots of beans. Regardless of the carbon source, T37 strain had significantly higher Sm1 expression and was chosen for further studies. When grown on different carbon sources, Sm1 expression was highest on galactose, bean seed, glucose and starch. Sm1 gene from T37 strain was cloned; it had a single exon, and encoded a protein of 138 amino acids, showing high sequence identity with some proteins belonging to the cerato-platanin family.

Effects of the Addition of Trichoderma reesei Cellulase to Broiler Chicken Diets for a 21-Day Period.

The cellulose present in the cell wall of vegetables prevents the greater release of nutrients to the animal. Therefore, the use of the cellulase enzyme is a viable strategy as it is capable of breaking cellulose bonds, releasing nutrients such as glucose, increasing dietary energy, and thus improving the productive performance of birds. Trichoderma reesei is efficient in the production of cellulase, which is produced via submerged fermentation followed by purification, formulation, and drying. Therefore, an experiment was carried out using 240 male broilers of the Cobb-500® lineage to verify the effects resulting from the addition of powdered (500 g/t and 1000 g/t) and liquid (500 mL/t) cellulase over a period of 1 to 21 days. A completely randomized experimental design was used, consisting of four treatments with six replications and ten birds per replication that were housed in an experimental cage. It was observed that performance and digestibility results were significantly different with cellulase supplementation. Also, the relative weight of the large intestine in the period between one and seven days increased when cellulase was added at 1000 g/t. In the period of between eight and 14 days of life, the birds that consumed only the basal diet obtained higher levels of liver protein than those that received the treatments with the addition of the enzyme. However, 15 and 21 days, the consumed feed effect did not occur between thus, it is not conclusive whether hepatotoxicity occurs with the addition of cellulase. For the blood parameters, at 21 days, the diets with added cellulase were not significantly different regarding electrolytes. It was concluded that this cellulase produced by Trichoderma reesei can be included in the animals' diet.

Identification of differentially expressed genes from Trichoderma harzianum during growth on cell wall of Fusarium solani as a tool for biotechnological application.

BACKGROUND: The species of T. harzianum are well known for their biocontrol activity against many plant pathogens. However, there is a lack of studies concerning its use as a biological control agent against F. solani, a pathogen involved in several crop diseases. In this study, we have used subtractive library hybridization (SSH) and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum genes expression during growth on cell wall of F. solani (FSCW) or glucose. RT-qPCR was also used to examine the regulation of 18 genes, potentially involved in biocontrol, during confrontation between T. harzianum and F. solani. RESULTS: Data obtained from two subtractive libraries were compared after annotation using the Blast2GO suite. A total of 417 and 78 readable EST sequence were annotated in the FSCW and glucose libraries, respectively. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on FSCW or glucose. We identified various genes of biotechnological value encoding to proteins which function such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. Fifteen genes were up-regulated and sixteen were down-regulated at least at one-time point during growth of T. harzianum in FSCW. During the confrontation assay most of the genes were up-regulated, mainly after contact, when the interaction has been established. CONCLUSIONS: This study demonstrates that T. harzianum expressed different genes when grown on FSCW compared to glucose. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against F. solani. The identification and evaluation of these genes may contribute to the development of an efficient biological control agent.

Mycoparasitism studies of Trichoderma species against three phytopathogenic fungi: evaluation of antagonism and hydrolytic enzyme production.

Trichoderma spp. are used for biocontrol of several plant pathogens. However, their efficient interaction with the host needs to be accompanied by production of secondary metabolites and cell wall-degrading enzymes. Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum. Trichoderma harzianum and T. asperellum were the most effective antagonists against the pathogens. Most of the Trichoderma species produced toxic volatile metabolites, having significant effects on growth and development of the plant pathogens. When these species were grown in liquid cultures with cell walls from these plant pathogens, they produced and secreted ß-1,3-glucanase, NAGAse, chitinase, acid phosphatase, acid proteases and alginate lyase.

Oral Treatment with Angiotensin-(1-7) Attenuates the Kidney Injury Induced by Gentamicin in Wistar Rats.

BACKGROUND: Acute Kidney Injury (AKI), a common disease of the urinary system, can be induced by high doses of gentamicin (GM). The renin-angiotensin system exerts a key role in the progression of the AKI since elevated intrarenal levels of Ang II, and ACE activity is found in this condition. However, it is unknown whether oral administration of angiotensin (Ang)-(1-7), a heptapeptide that evokes opposite effects of Ang II, may attenuate the renal injuries induced by gentamicin. OBJECTIVES: To evaluate the effects of Ang-(1-7) on GM-induced renal dysfunction in rats. METHODS: AKI was induced by subcutaneous administration of GM (80 mg/Kg) for 5 days. Simultaneously, Ang-(1-7) included in hydroxypropyl ß-cyclodextrin (HPßCD) was administered by gavage [46 µg/kg HPßCD + 30 µg/kg Ang-(1-7)]. At the end of the treatment period (sixth day), the rats were housed in metabolic cages for renal function evaluation. Thereafter, blood and kidney samples were collected. RESULTS: Ang-(1-7) attenuated the increase of the plasmatic creatinine and proteinuria caused by GM but did not change the glomerular filtration rate nor tubular necrosis. Ang-(1-7) attenuated the increased urinary flow and the fractional excretion of H2O and potassium observed in GM rats but intensified the elevated excretion of sodium in these animals. Morphological analysis showed that Ang-(1-7) also reduced the tubular vacuolization in kidneys from GM rats. CONCLUSION: Ang-(1-7) promotes selective beneficial effects in renal injuries induced by GM.

New insights in Trichoderma harzianum antagonism of fungal plant pathogens by secreted protein analysis.

Trichoderma harzianum ALL42 were capable of overgrowing and degrading Rhizoctonia solani and Macrophomina phaseolina mycelia, coiling around the hyphae with formation of apressoria and hook-like structures. Hyphae of T. harzianum ALL42 did not show any coiling around Fusarium sp. hyphae suggesting that mycoparasitism may be different among the plant pathogens. In this study, a secretome analysis was used to identify some extracellular proteins secreted by T. harzianum ALL42 after growth on cell wall of M. phaseolina, Fusarium sp., and R. solani. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. A total of 60 T. harzianum ALL42 secreted proteins excised from the gel were analyzed from the three growth conditions. While seven cell wall-induced proteins were identified, more than 53 proteins spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced. Endochitinase, ß-glucosidase, α-mannosidase, acid phosphatase, α-1,3-glucanase, and proteases were identified in the gel and also detected in the supernatant of culture.

Anatomical changes induced by isolates of Trichoderma spp. in soybean plants.

In the current work we evaluated the anatomical changes induced by T. harzianum and T. asperellum in two soybean cultivars, BRSGO Caiaponia and NA 5909 RG. Soybean production represents a growing market worldwide, and new methods aimed at increasing its productivity and yield are constantly being sought. Fungi of the genus Trichoderma have been widely used in agriculture as a promising alternative for the promotion of plant growth and for biological control of various pathogens. It is known that Trichoderma spp. colonize plant roots, but the anatomical changes that this fungus can cause are still less studied. Experiment was conducted in a greenhouse to collect leaves and soybean roots to perform analysis of growth parameters, enzymatic activity of defense-related enzymes and anatomical changes. It was observed that inoculation of Trichoderma spp. caused anatomical alterations, among them, increase in stomatal index at the abaxial leaf surface, thickness of the root cortex, thickness of adaxial epidermis, mean diameter of the vascular cylinder, thickness of the mesophyll, and thickness of the spongy parenchyma of the soybean plants. These results indicate that the alterations in these factors may be related to the process of plant resistance to pathogens, and better performance against adverse conditions. This study demonstrates that the anatomical study of plants is an important tool to show the effects that are induced by biological control agents.

The boosting effect of recombinant hemicellulases on the enzymatic hydrolysis of steam-treated sugarcane bagasse.

To increase the efficiency of enzyme cocktails in deconstructing cellulose and hemicelluloses present in the plant cell wall, a combination of enzymes with complementary activities is required. Xylan is the main hemicellulose component of energy crops and for its complete hydrolysis a system consisting of several enzymes acting cooperatively, including endoxylanases (XYN), ß-xylosidases (XYL) and α-l-arabinofuranosidases (ABF) is necessary. The current work aimed at evaluating the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of steam-exploded sugarcane bagasse (SEB). One recombinant endoxylanase (HXYN2) and one recombinant ß-xylosidase (HXYLA) from Humicola grisea var thermoidea, together with an α-l-arabinofuranosidase (AFB3) from Penicillium pupurogenum, all produced in Pichia pastoris, were used to formulate an efficient enzyme mixture for SEB hydrolysis using a 23 Central Composite Rotatable Design (CCRD). The most potent enzyme for SEB hydrolysis was ABF3. Subsequently, the optimal enzyme mixture was used in combination with commercial cellulases (Accellerase 1500), either simultaneously or in sequential experiments. The supplementation of Accellerase 1500 with hemicellulases enhanced the glucose yield from SEB hydrolysis by 14.6%, but this effect could be raised to 50% when hemicellulases were added prior to hydrolysis with commercial cellulases. These results were supported by scanning electron microscopy, which revealed the effect of enzymatic hydrolysis on SEB fibers. Our results show the potential of complementary enzyme activities to improve enzymatic hydrolysis of SEB, thus improving the efficiency of the hydrolytic process.

Involvement of G-alpha protein GNA3 in production of cell wall-degrading enzymes by Trichoderma reesei (Hypocrea jecorina) during mycoparasitism against Pythium ultimum.

The involvement of the G-alpha protein GNA3 in the production of cell wall-degrading enzymes (CWDEs) by Trichoderma reesei during antagonism against Pythium ultimum was investigated. cAMP content was 2.8-fold higher in the T. reesei mutant gna3QL than in the parental TU-6. The gna3QL, like TU-6, inhibited the growth of P. ultimum in dual culture assays. Scanning electron microscopy showed that the gna3QL promoted more morphological alterations of P. ultimum cell wall than TU-6. In general, gna3QL produced higher activities of CWDEs than TU-6. We therefore suggest that CWDEs production during mycoparasitism by T. reesei against P. ultimum may be associated with the level of GNA3 activity.

Morphological and molecular aspects of sclerotial development in the phytopathogenic fungus Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary produces a resistance structure called sclerotium, which guarantees its survival in soil for long periods. Morphological and melanization aspects during sclerotial development were evaluated by microscopy and qRT-PCR techniques. S. sclerotiorum produces sclerotia with different phases of maturation and melanization during growth in PDA medium. Using scanning electron microscopy we observed that there are no structural differences in the three stages of formation of melanized and non-melanized sclerotium. Through histochemical analysis we observed that the melanized sclerotium accumulates more glycogen and produces less protein than non-melanized sclerotia. Melanin was most commonly found in the rind of melanized sclerotia, and the highest concentration of lipofucsins was found in non-melanized sclerotia. These molecules are products of the lipid peroxidation pathway and are associated with oxidative stress during differentiation in fungi. The expression of histidine kinase (shk) and adenylate cyclase (sac) genes in melanized and non-melanized sclerotiawere also evaluated. The higher gene expression of shk and lesser expression of sac in non-melanized sclerotiais an indication of the participation of cell signaling in the development of these structures. The higher expression of polyketide synthase (pks), tyrosinase (tyr) and laccase (lac) in non-melanized sclerotia suggested that S. sclerotiorum can use the DHN and L-dopa pathways to produce melanin. Expression studies of the enzymes chitin synthase and glucan synthase suggest that this process occurs along with the formation of melanin. This is interesting since polysaccharides, such as chitin and ß-1,3-glucan, serve as a scaffold to which the melanin granules are cross-linked.

Hsp genes are differentially expressed during Trichoderma asperellum self-recognition, mycoparasitism and thermal stress.

Heat shock proteins (Hsp) are important factors in the response of organisms to oscillations in environmental conditions. Although Hsp have been studied for a long time, little is known about this protein class in Trichoderma species. Here we studied the expression of Hsp genes during T. asperellum growth, and mycoparasitism against two phytopathogens: Sclerotinia sclerotiorum and Fusarium oxysporum, as well as during thermal stress. The expression levels of these genes were observed by real-time PCR and they showed to be differentially expressed under these conditions. We verified that the TaHsp26c, TaHsp70b and TaHsp70c genes were differentially expressed over time, indicating that these genes can be developmentally regulated in T. asperellum. Except for TaHsp26a, all other genes analyzed were induced in the post-contact condition when T. asperellum was cultured in a confrontation plate assay against itself. Additionally, TaHsp26b, TaHsp26c, TaHsp90, TaHsp104a and TaHsp104b were induced during initial contact between T. asperellum hyphae, suggesting that these proteins must play a role in the organism´s self-recognition mechanism. When we examined gene expression during mycoparasitism, we observed that some genes were induced both by S. sclerotiorum and F. oxysporum, while others were not induced during interaction with either of the phytopathogens. Furthermore, we observed some genes induced only during confrontation against S. sclerotiorum, indicating that the expression of Hsp genes during mycoparasitism seems to be modulated by the phytopathogen. To assess whether such genes are expressed during temperature oscillations, we analyzed their transcription levels during thermal and cold shock. We observed that except for the TaHsp70c gene, all others presented high transcript levels when T. asperellum was submitted to high temperature (38 °C), indicating their importance in the response to heat stress. The TaHsp70c gene was significantly induced only in cold shock at 4 °C. Our results show the importance of Hsp proteins during self-recognition, mycoparasitism and thermal stress in T. asperellum.

Biochemical and Molecular Study of Trichoderma harzianum Enriched Secretome Protein Profiles Using Lectin Affinity Chromatography.

Protein glycosylation is one of the most studied post-translational modifications and has received considerable attention for its critical role in the cell biology of eukaryotic cells. The genus Trichoderma has been extensively studied in the biocontrol of soil-borne fungal phytopathogens. The aim of this study was to identify the proteins secreted from Trichoderma harzianum after interacting with the cell walls of two phytopathogens, Sclerotinia sclerotiorum and Fusarium oxysporum. This study used N-glycoprotein enrichment with a concanavalin A (Con A) affinity column, staining detection differential SDS-PAGE, sequencing by mass spectrometric, and protein identification by comparison with the NCBI database to detect the protein expression of the two resulting secretome samples. The majority of the proteins found in both enriched secretomes belonged to a specific class of carbohydrate-active enzymes (CAZymes), within which glycosyl hydrolases (GHs), glycosyltransferases (GTs), and auxiliary activities (AAs) were identified. In this study was described two proteins that have not been previously reported in the secretomes of Trichoderma, a glycosyltransferase (six-harpin) and a galactose oxidase, belonging to the class of auxiliary activities (AA), classified as an AA subfamily AA5-2.The expression pattern of gene encoding to 17 identified proteins, evaluated by real-time quantitative PCR (RT-qPCR), showed significant difference of expression of some GHs and proteases, suggesting a specific gene expression regulation by T. harzianum in presence of different cell walls of two phytopathogens.

Endo-ß-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens.

Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ß-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ß-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-ß-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.