Matteo Lorito - Academia.edu (original) (raw)

Papers by Matteo Lorito

Journal of bacteriology, 1996

We have investigated the molecular basis for the reported synergism between peptaibols and cell w... more We have investigated the molecular basis for the reported synergism between peptaibols and cell wall hydrolytic enzymes in the antagonism of phytopathogenic fungi by Trichoderma harzianum. beta-Glucan synthase activity on isolated plasma membranes of Botrytis cinerea was inhibited in vitro by the peptaibols trichorzianin TA and TB, and this inhibition was reversed by the addition of phosphatidylcholine. beta-Glucan synthesis in vivo, assayed by the incorporation of [2-(3)H]glucose into cell wall material, was inhibited by the presence of peptaibols, and this inhibition was synergistic with exogenously added T. harzianum beta-1,3-glucanase. This synergism is therefore explained by an inhibition of the membrane-bound beta-1,3-glucan synthase of the host by the peptaibols, which inhibit the resynthesis of cell wall beta-glucans, sustain the disruptive action of beta-glucanases, and all together enhance the fungicidal activity. Therefore, we have identified cell wall turnover as a major...

Current genetics, 1993

Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for ... more Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for transforming strains of Gliocladium virens and Trichoderma harzianum. For biolistic transformation, conidia were bombarded using a helium-driven biolistic device to accelerate M5 tungsten particles coated with plasmid or genomic DNA. DNA from either source contained a bacterial hygromycin B resistance gene (hygB) as a dominant selectable marker. The same sources of DNA were also used to transform protoplasts using a standard polyethylene glycol-CaCl2 protoplast fusion protocol. Hygromycin B-resistant (HygBR) transformants were recovered from all strains, methods, and DNA sources except for genomic DNA used with the protoplast method. The biolistic procedure was technically simpler, and increased transformation frequency and genetic stability in the progeny as compared with the protoplast-mediated transformation. Southern analysis of homokaryotic HygBR progenies showed that the transformi...

Journal of Zhejiang University SCIENCE, 2004

Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from... more Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.

Transgenic Research, 2008

Constitutive over-expression of antifungal genes from microorganisms involved in plant defence me... more Constitutive over-expression of antifungal genes from microorganisms involved in plant defence mechanisms represents a promising strategy for conferring genetic resistance against a broad range of plant pathogenic fungi. In the present work, two transgenic lemon clones with the chit42 gene from Trichoderma harzianum were tested for resistance to fungal disease and expression level of defence-related genes was evaluated. Different resistance-related processes, such as production of reactive oxygen species (ROS), systemic acquired resistance (SAR) and induced systemic resistance (ISR), were monitored in transgenic and wild type lemon clones inoculated with Botrytis cinerea, the causal agent of grey mould in citrus. Expression of genes that encode gluthatione peroxidase (GPX), a producer of ROS, chitinases, glucanases (SAR), PAL, HPL, and AOS (ISR) was measured by quantitative PCR during the first 24 h after leaf inoculation. Leaves of transgenic lemon plants inoculated with B. cinerea showed significantly less lesion development than wild type leaves. Tissues from detached leaves of different transgenic lemon clones showed a significant correlation between resistance and transgene expression. On the other hand, the over-expression of the transgenic fungal gene enhanced by two-three folds transcript levels of genes associated with enhanced ROS production and ISR establishment, while the expression of native chitinase and glucanase genes involved in SAR was down-regulated.

Soil Biology and Biochemistry, 2009

Accumulation of rare earth elements (REE) in the soil may be due to the use of REE enriched ferti... more Accumulation of rare earth elements (REE) in the soil may be due to the use of REE enriched fertilizers and to contamination by REE containing wastes. Although widely used in China for soil and foliar dressing of crops, little is known about the effect of REE applications on the soil microbial community. The effect of REE on the growth of biological control strains of Trichoderma atroviride and Trichoderma harzianum was investigated in vitro using either a mix of different REE containing different amounts of lanthanum, cerium, praseodymium, neodymium, gadolinium nitrate and lanthanum nitrate alone in comparison to treatments with potassium nitrate and water. In plate tests applied concentrations ranged from 0.1 mM to 300 mM for lanthanum and REE mix and from 0.1 mM to 900 mM for the potassium solution. In liquid culture tests applied concentrations ranged from 0.001 mM to 100 mM for lanthanum and REE mix and from 0.003 mM to 900 mM for the potassium solution. ICP-MS, TEM and TEM X-ray microanalysis were used to study the accumulation of REE in fungal biomass. All the Trichoderma strains showed a good tolerance to the presence of REE in the culture media. Some growth enhancing effects were observed in liquid cultures of T. harzianum strains but not in T. atroviride. Accumulation of REE in fungal biomass, both at intracellular level and in the extracellular matrix, was observed.

Proceedings of the National Academy of Sciences, 1996

The fungus Trichoderma harzianum is a potent mycoparasite of various plant pathogenic fungi. We h... more The fungus Trichoderma harzianum is a potent mycoparasite of various plant pathogenic fungi. We have studied the molecular regulation of mycoparasitism in the host͞mycoparasite system Botrytis cinerea͞T. harzianum. Protein extracts, prepared from various stages of mycoparasitism, were used in electrophoretic mobility-shift assays (EM-SAs) with two promoter fragments of the ech-42 (42-kDa endochitinase-encoding) gene of T. harzianum. This gene was chosen as a model because its expression is triggered during mycoparasitic interaction [Carsolio, C., Gutierrez, A., Jimenez, B., van Montagu, M. & Herrera-Estrella, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10903-10907].

Proceedings of the National Academy of Sciences, 1998

Disease resistance in transgenic plants has been improved, for the first time, by the insertion o... more Disease resistance in transgenic plants has been improved, for the first time, by the insertion of a gene from a biocontrol fungus. The gene encoding a strongly antifungal endochitinase from the mycoparasitic fungus Trichoderma harzianum was transferred to tobacco and potato. High expression levels of the fungal gene were obtained in different plant tissues, which had no visible effect on plant growth and development. Substantial differences in endochitinase activity were detected among transformants. Selected transgenic lines were highly tolerant or completely resistant to the foliar pathogens Alternaria alternata, A. solani, Botrytis cinerea, and the soilborne pathogen Rhizoctonia solani. The high level and the broad spectrum of resistance obtained with a single chitinase gene from Trichoderma overcome the limited efficacy of transgenic expression in plants of chitinase genes from plants and bacteria. These results demonstrate a rich source of genes from biocontrol fungi that can be used to control diseases in plants.

Plant Disease, 2010

Page 1. 928 Plant Disease / Vol. 94 No. 8 Changing Models for Commercialization and Implementatio... more Page 1. 928 Plant Disease / Vol. 94 No. 8 Changing Models for Commercialization and Implementation of Biocontrol in the Developing and the Developed World Microbially based biocontrol of plant diseases differs fundamen ...

Plant Breeding, 2007

Antifungal genes from micro-organisms were inserted into the genome of a number of plant species,... more Antifungal genes from micro-organisms were inserted into the genome of a number of plant species, representing a promising strategy for conferring genetic disease resistance against a broad range of plant pathogenic fungi. In the present study, the chit42 gene from Trichoderma harzianum (codifying the antifungal protein endochitinase) was introduced into the ÔFemminello siracusanoÕ lemon by Agrobacterium tumefaciens, in order to regenerate transgenic plants resistant to fungal disease. Three polymerase chain reaction (PCR)-positive clones were obtained. Southern blot confirmed the integration of the transgene in the lemon genome and revealed that one or two copies had been inserted. Reverse transcriptase-PCR, Northern blot and Western analysis were performed and the results confirmed the expression of the inserted gene. The transgenic clones were tested in vitro and in vivo for disease resistance. Conidia germination and fungal growth of Phoma tracheiphila were strongly inhibited in vitro by the transgenic foliar proteins, while no effects were observed with the controls. Disease resistance assays were performed in vivo with Botrytis cinerea, the causal agent of grey mould in fruit. Transgenic lemon plants, inoculated with lemon petals infected by a single-conidial isolate of B. cinerea, showed significantly less lesion development than the controls. On the whole, the results indicate that the transformation with the antifungal endochitinase gene may represent a strategy for disease control in citrus crops.

Phytopathology, 1993

Two chitinolytic enzymes from Trichoderma harzianum strain P1 were tested for their antifungal ac... more Two chitinolytic enzymes from Trichoderma harzianum strain P1 were tested for their antifungal activity in bioassays against nine different fungal species. Spore germination (or cell replication) and germ tube elongation were inhibited for all chitin-containing fungi ...

Molecular Plant-Microbe Interactions, 2009

Considering the complexity of the in vivo interactions established by a mycoparasitic biocontrol ... more Considering the complexity of the in vivo interactions established by a mycoparasitic biocontrol agent at the plant rhizosphere, proteomic, genomic, and transcriptomic approaches were used to study a novel Trichoderma gene coding for a plant cell wall (PCW)-degrading enzyme. A proteome analysis, using a three-component (Trichoderma spp.-tomato plantlets-pathogen) system, allowed us to identify a differentially expressed Trichoderma harzianum endopolygalacturonase (endoPG). Spot 0303 remarkably increased only in the presence of the soilborne pathogens Rhizoctonia solani and Pythium ultimum, and corresponded to an expressed sequence tag from a T. harzianum T34 cDNA library that was constructed in the presence of PCW polymers and used to isolate the Thpg1 gene. Compared with the wild-type strain, Thpg1-silenced transformants showed lower PG activity, less growth on pectin medium, and reduced capability to colonize tomato roots. These results were combined with microarray comparative data from the transcriptome of Arabidopsis plants inoculated with the wild type or a Thpg1-silenced transformant (ePG5). The endoPG-encoding gene was found to be required for active root colonization and plant defense induction by T. harzianum T34. In vivo assays showed that Botrytis cinerea leaf necrotic lesions were slightly smaller in plants colonized by ePG5, although no statistically significant differences were observed.

Molecular Biotechnology, 1994

Fungal cell wall degrading enzymes produced by the biocontrol fungi Trichoderma harzianum and Gli... more Fungal cell wall degrading enzymes produced by the biocontrol fungi Trichoderma harzianum and Gliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes from T. harzianum were able to improve substantially the antifungal ability of a biocontrol strain of Enterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library of T. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.

Microbiology, 1994

Different classes of cell wall degrading enzymes produced b y the biocontrol fungi Trichoderma ha... more Different classes of cell wall degrading enzymes produced b y the biocontrol fungi Trichoderma harzianum and Gliocladium virens inhibited spore germination of Botrytis cinerea in a bioassay in vitro. The addition of any chitinolytic or glucanolytic enzyme to the reaction mixture synergistically enhanced the antifungal properties of five different fungitoxic compounds against B. cinerea. The chemicals tested were gliotoxin, flusilazole, miconazole, captan and benomyl. Dose response curves were determined for each combination of toxin and enzyme, and in all cases the ED, , values of the mixtures were substantially lower than ED, , values of the two compounds used alone. For instance, the addition of endochitinase from T. harzianum at a concentration of 10 pg ml level of synergism appeared to be higher when enzymes were combined with toxins having primary sites of action associated with membrane structure, compared with pesticides having multiple or cytoplasmic sites of action. Among enzymes tested, the highest levels of synergism with synthetic fungicides were detected for the endochitinase from T. harzianum strain P I , which, when used alone, was the most effective chitinolytic enzyme against phytopathogenic fungi of those tested. The use of hydrolytic enzymes to synergistically enhance the antifungal ability of fungitoxic compounds may reduce the impact of some chemical pesticides on plants and animals. reduced the ED, , values of toxins up to 86-fold. The

Letters in Applied Microbiology, 2006

Aims: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in ... more Aims: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. Methods and Results: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. Conclusions: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. Significance and Impact of the Study: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.

Letters in Applied Microbiology, 2010

Journal of Natural Products, 2009

A Trichoderma harzianum strain, isolated from composted hardwood bark in Western Australia, was f... more A Trichoderma harzianum strain, isolated from composted hardwood bark in Western Australia, was found to produce a metabolite with antifungal and plant growth promoting activity. The structure and absolute configuration of the fungal compound, harzianic acid (1), were determined by X-ray diffraction studies. Harzianic acid showed antibiotic activity against Pythium irregulare, Sclerotinia sclerotiorum, and Rhizoctonia solani. A plant growth promotion effect was observed at low concentrations of 1.

Journal of General Virology, 1989

Complementary DNA clones representing approximately 6100 nucleotides of potato leaf roll virus (P... more Complementary DNA clones representing approximately 6100 nucleotides of potato leaf roll virus (PLRV) were generated, restriction-mapped, and partially sequenced. Within one of the cDNA clones an open reading frame (ORF) encoding a 23K protein was identified and further characterized. Amino acid sequence comparison of this protein showed significant homology (47.1~) with the barley yellow dwarf virus (BYDV-PAV) coat protein. This and other observations suggested that this gene encodes the PLRV coat protein. Other similarities were observed between PLRV and BYDV sequences in this region of their genomes, including an ORF of 17K within the ORF encoding the 23K putative coat protein.

Journal of Agricultural and Food Chemistry, 2006

Nutritionists recommend increasing the intake of soluble dietary fiber (SDF), which is very low i... more Nutritionists recommend increasing the intake of soluble dietary fiber (SDF), which is very low in most cereal-based products. Conversion of insoluble DF (IDF) into SDF can be achieved by chemical treatments, but this affects the sensorial properties of the products. In this study, the possibility of getting a substantial increase of SDF from cereal products using a tailored preparation of Trichoderma enzymes is reported. Enzymes were produced cultivating Trichoderma using durum wheat fiber (DWF) and barley spent grain (BSG) as unique carbon sources. Many Trichoderma strains were screened, and the hydrolysis conditions able to increase by enzymatic treatment the amount of SDF in DWF and BSG were determined. Results demonstrate in both products that it is possible to triple the amount of SDF without a marked decrease of total DF. The enzymatic treatment also causes the release of hydroxycinnamic acids, mainly ferulic acid, that are linked to the polysaccharides chains. This increases the free phenolic concentration, the water-soluble antioxidant activity, and, in turn, the phenol compounds bioavailability.

Gene, 1996

A full-length cDNA close, Th1433, (GenBank accession No. U24158), was isolated and characterized ... more A full-length cDNA close, Th1433, (GenBank accession No. U24158), was isolated and characterized from the filamentous fungus, Trichoderma harzianum. The deduced amino acid (aa) sequence showed an acidic 30-kDa protein homologous to the 14-3-3 proteins, a family of putative kinase regulators originally characterized in mammalian brain tissue. The greatest homology, 71% identical aa, was found to BMH1, the corresponding protein from Saccharomyces cerevisiae and to the epsilon isoform from sheep brain. Southern analysis of genomic DNA indicated that Th1433 is a member of a small genomic family. At least two genes encoding 14-3-3-like proteins exist in T. harzianum. Northern analysis showed the highest level of expression during the first day after inoculation of the culture with conidial spores.

Gene, 1994

There are no reports of gene sequences coding for extracellular chitinolytic enzymes from filamen... more There are no reports of gene sequences coding for extracellular chitinolytic enzymes from filamentous fungi, even though these enzymes are considered critical to the biological control of plant pathogenic fungi. The purpose of this paper was to report the isolation of a gene (ThEn-42) encoding endochitinase (Ech) from Trichoderma harzianum strain P1, describe its sequence, and to determine whether it was related to genes coding for enzymes with similar functions from prokaryotic or other eukaryotic sources. A clone containing a 1096-bp foreign cDNA fragment was isolated from thalli grown under induced conditions. This cDNA molecule was sequenced and found to lack a portion of the 5' terminus. Polymerase chain reaction (PCR) was used to isolate a fragment from the lambda gt11 library which contained the 5' terminus plus an overlap region with the 1096-bp cDNA clone. The full-length cDNA sequence, consisting of 1554 bp, contained an open reading frame (ORF) expressing a protein of 424 amino acids (aa). Southern analysis of genomic DNA indicated that there is only a single gene in strain P1 with sequence identity to the sequence described in this report. One region within the protein, thought to be required for catalytic activity of the enzyme, was highly conserved between genes coding for Ech from Th, Serratia marcescens, Bacillus circulans, Streptomyces plicatus, Vibrio parahemolyticus and Kluyveromyces lactis.