Biomedical subjects

I Chet

Publications and source records attributed to I Chet.

At least 19 recordsLinked to original sources

PubMed2005

Mutual relationships between soils and biological carrier systems.

Improved viability and antagonistic activity of biocontrol agents during soil inoculation is of crucial importance to their effective application. The chitinolytic bacterium Serratia marcescens was used as a model organism to study the efficacy of freeze-dried alginate beads (in comparison to their non-dried counterparts) as possible carriers for immobilized biocontrol agents. The release of bacteria and chitinolytic enzyme from alginate beads, before and during their application in soil, was examined, and the beads' physical properties characterized. Dispersal of the alginate bead-entrapped S. marcescens in the soil resulted in high soil cell densities throughout the 35 days of the experiment. Chitin inclusion in the beads resulted in significantly higher chitinolytic activity of S. marcescens, increased dry-bead porosity and decreased stiffness. Rehydration of the dried beads (after immersion in soil) resulted in a sixfold increase in weight due to water absorption. No significant differences were found in bacterial count inside the non-dried (gel) versus dried beads. However, higher cell densities and chitinase activity were detected in soil containing dried beads with chitin than in that containing their non-dried counterparts. The biological performance of S. marcescens was examined in the greenhouse: a free cell suspension reduced bean (Phaseolus vulgaris L.) disease by 10%, while immobilized bacteria found in the dried, chitin-containing beads reduced disease by 60%.

Alginates↗

PubMed2005

Activity of Serratia plymuthica IC1270 gene chiA promoter region in Escherichia coli mutants deficient in global regulators of transcription.

To study the regulation of expression of the Serratia plymuthica gene chiA encoding a 58-kDa endochitinase, its 586-bp-long upstream regulatory region was cloned, sequenced and fused to a promoterless lac operon in phage lambdaRS45 to obtain a single-copy transcriptional fusion (P F1chiA )-lac in lysogens of Escherichia coli wild-type strains or their mutants deficient in various global regulators of transcription. The level of P F1chiA -lac expression increased about 20- and 90-fold, respectively, in E. coli K12 Deltahns and double Deltahns stpA mutants deficient in H-NS, and in both H-NS and StpA DNA-binding histone-like proteins, as compared to levels in the wild-type strain. In a Deltalrp mutant deficient in the leucine-responsive transcriptional regulator Lrp, the level of P F1chiA -lac expression increased only up to threefold, whereas even smaller differences relative to the wild-type strain were observed in rpoS and Deltacrp mutants deficient in the sigmaS subunit of RNA polymerase and catabolite-repression protein (CRP), respectively. Deletion of the inverted-repeat sequences and curved DNA regions located in the upstream region of chiA essentially did not influence strain IC1270's chiA promoter activity in E. coli .

Base Sequence↗

PubMed2004

Unexpected distribution of immobilized microorganisms within alginate beads.

Immobilization refers to the prevention of free cell movement by natural or artificial means. It has always been assumed that immediately after an immobilization procedure is performed, cells are distributed homogeneously in the beads that entrap them. However, in this study, Escherichia coli and Trichoderma asperellum distribution in alginate-gel beads was found to be nonhomogeneous. In fact, there was a greater presence of cells on the surface of the alginate beads than in their cores.

Alginates↗

PubMed2003

Structure of dried cellular alginate matrix containing fillers provides extra protection for microorganisms against UVC radiation.

Soil microorganisms in general and biocontrol agents in particular are very sensitive to UV light. The packaging of biocontrol microorganisms into cellular solids has been developed as a means of reducing loss caused by exposure to environmental UV radiation. The bacterial and fungal biocontrol agents Pantoea agglomerans and Trichoderma harzianum were immobilized in freeze-dried alginate beads containing fillers and subjected to 254 nm UV radiation (UVC). Immobilization of cells in freeze-dried alginate-glycerol beads resulted in greater survival after UV irradiation than for a free cell suspension. Adding chitin, bentonite or kaolin as fillers to the alginate-glycerol formulation significantly increased bacterial survival. Immobilization in alginate-glycerol-kaolin beads resulted in the highest levels of survival. The transmissive properties of the dried hydrocolloid cellular solid had a major influence on the amount of protection by the cell carrier. Dried alginate matrix (control) transmitted an average of 7.2% of the radiation. Filler incorporation into the matrix significantly reduced UV transmission: Alginate with kaolin, bentonite and chitin transmitted an average of 0.15, 0.38 and 3.4% of the radiation, respectively. In addition, the filler inclusion had a considerable effect on the bead's average wall thickness, resulting in a approximately 1.5- to threefold increase relative to beads based solely on alginate. These results suggest that the degree of protection of entrapped microorganisms against UVC radiation is determined by the UV-transmission properties of the dried matrix and the cellular solid's structure. It is concluded that for maximum protection against UV-radiation-induced cell loss, biocontrol microorganisms should be immobilized in alginate-glycerol beads containing kaolin.

Alginates↗

PubMed2001

Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM.

A novel 36-kDa endochitinase named chit36 has been isolated and characterized from Trichoderma harzianum Rifai TM. Partial amino acid sequences from the purified protein were used to clone the fungal cDNA, based on polymerase chain reaction with degenerate primers. The complete open reading frame encodes a 344-amino acid protein which shows 84% similarity to a putative chitinase from Streptomyces coelicolor. Chit36 was overexpressed under the pki1 constitutive promoter from Trichoderma reesei via biolistic transformation of T. harzianum TM. Stable transformants showed expression and endochitinase activity of chit36 in glucose-rich medium. Culture filtrates containing secreted CHIT36 as the sole chitinolytic enzyme completely inhibited the germination of Botrytis cinerea conidia. Growth of Fusarium oxysporum f. sp. melonis and Sclerotium rolfsii were significantly inhibited on agar plates on which the Trichoderma transformants had previously been grown.

Amino Acid Sequence↗

PubMed2001

A convenient fluorometric method for the detection of extracellular N-acetylglucosaminidase production by filamentous fungi.

A rapid and convenient method for the detection of chitinases accumulating in filamentous fungal cultures was developed. The assay is performed on cultures growing in microtiter plates, with a fluorogenic substrate: 4-methylumbelliferyl-N-acetyl-D-glucosaminide (4-MeUNAG). The fluorescence of the product, 4-methylumbelliferone, was detected. This method was successfully used to follow induction and repression of extracellular exochitinase activity in the biocontrol fungus Trichoderma harzianum.

Acetylglucosaminidase↗

PubMed2001

The defense response elicited by the pathogen Rhizoctonia solani is suppressed by colonization of the AM-fungus Glomus intraradices.

Defense responses of alfalfa roots to the pathogenic fungus Rhizoctonia solani were reduced significantly in roots simultaneously infected with the vesicular arbuscular mycorrhizal (AM) fungus Glomus intraradices. R. solani induced five- to tenfold increases in the steady-state levels of chalcone isomerase and isoflavone reductase mRNAs a doubling of root peroxidase activity and a marked autofluorescence in the infected tissue. These changes were inhibited by the presence of G. intraradices. Interestingly, germination of G. intraradices spores and hyphal elongation were sensitive to low concentrations (2 µM) of medicarpin-3-O-glucoside, an isoflavonoid phytoalexin that accumulated both in roots colonized by the pathogenic fungus as well as in AM-treated roots receiving high P, where no colonization by the beneficial fungus occurred. These data support the hypothesis that during early stages of colonization by G. intraradices, suppression of defense-related properties is associated with the successful establishment of AM symbiosis.

Journal Article↗

PubMed1999

Molecular characterization of a novel beta-1,3-exoglucanase related to mycoparasitism of Trichoderma harzianum.

Trichoderma harzianum, a soil-borne filamentous fungus, is capable of parasitizing several plant pathogenic fungi. Secretion of lytic enzymes, mainly glucanases and chitinases, is considered the most crucial step of the mycoparasitic process. The lytic enzymes degrade the cell walls of the pathogenic fungi, enabling Trichoderma to utilize both their cell walls and cellular contents for nutrition. We have purified a 110kDa novel extracellular beta-1,3-exoglucanase from T. harzianum, grown with laminarin or in dual cultures with host fungi. The corresponding gene, lam1.3, and its cDNA were isolated and their nucleotide sequences determined. The deduced amino-acid sequence predicted a molecular mass of 110.7kDa of a mature protein excluding a signal peptide. LAM1.3 showed high homology to EXG1, a beta-1,3-exoglucanase of the phytopathogenic fungus Cochliobolus carbonum, and a lower homology to BGN13.1, a beta-1,3-endoglucanase isolated from T. harzianum. However, it contains a unique C-terminal embodying cysteine motifs. The expression of lam1.3 in growth with laminarin, but not with glucose, was found to be a result of differential accumulation of the corresponding mRNA.

Amino Acid Sequence↗

PubMed1999

Chitinases in biological control.

The public concern over the harmful effects of chemical pesticides on the environment and human health has enhanced the search for safer, environmentally friendly control alternatives. Control of plant pests by the application of biological agents holds great promise as an alternative to the use of chemicals. It is generally recognized that biological control agents are safer and more environmentally sound than is reliance on the use of high volumes of pesticides. Due to the importance of chitinolytic enzymes in insect, nematode, and fungal growth and development, they are receiving attention in regard to their development as biopesticides or chemical defense proteins in transgenic plants and microbial biocontrol agents. In this sense, biological control of some soil-borne fungal diseases has been correlated with chitinase production. Fungi- and bacteria-producing chitinases exhibit antagonism against fungi, and inhibition of fungal growth by plant chitinases has been demonstrated. Insect pathogenic fungi have considerable potential for the biological control of insect pests. Entomopathogenic fungi apparently overcome physical barriers of the host by producing multiple extracellular enzymes including chitinolytic enzymes, which help to penetrate the cuticle and facilitate infection. In this chapter, the role of chitinases in biological control and their potential use in the improvement of biocontrol agents and crop plants by genetic engineering is analyzed in view of recent findings.

Bacterial Physiological Phenomena↗

PubMed1999

Mycorrhiza-induced changes in disease severity and PR protein expression in tobacco leaves

The development of leaf disease symptoms and the accumulation of pathogenesis-related (PR) proteins were monitored in leaves of tobacco (Nicotiana tabacum cv. Xanthinc) plants colonized by the arbuscular mycorrhizal fungus Glomus intraradices. Leaves of mycorrhizal plants infected with the leaf pathogens Botrytis cinerea or tobacco mosaic virus showed a higher incidence and severity of necrotic lesions than those of nonmycorrhizal controls. Similar plant responses were obtained at both low (0.1 mM) and high (1.0 mM) nutritional P levels and with mutant plants (NahG) that are unable to accumulate salicylic acid. Application of PR-protein activators induced PR-1 and PR-3 expression in leaves of both nonmycorrhizal and mycorrhizal plants; however, accumulation and mRNA steady-site levels of these proteins were lower, and their appearance delayed, in leaves of the mycorrhizal plants. Application of 0.3 mM phosphate to the plants did not mimic the delay in PR expression observed in the mycorrhizal tobacco. Together, these data strongly support the existence of regulatory processes, initiated in the roots of mycorrhizal plants, that modify disease-symptom development and gene expression in their leaves.

Journal Article↗

PubMed1999

Induction of defense responses in cucumber plants (Cucumis sativus L. ) By the biocontrol agent trichoderma harzianum

The potential of the biocontrol agent Trichoderma harzianum T-203 to trigger plant defense responses was investigated by inoculating roots of cucumber seedlings with Trichoderma in an aseptic, hydroponic system. Trichoderma-treated plants were more developed than nontreated plants throughout the experiment. Electron microscopy of ultrathin sections from Trichoderma-treated roots revealed penetration of Trichoderma into the roots, restricted mainly to the epidermis and outer cortex. Strengthening of the epidermal and cortical cell walls was observed, as was the deposition of newly formed barriers. These typical host reactions were found beyond the sites of potential fungal penetration. Wall appositions contained large amounts of callose and infiltrations of cellulose. The wall-bound chitin in Trichoderma hyphae was preserved, even when the hyphae had undergone substantial disorganization. Biochemical analyses revealed that inoculation with Trichoderma initiated increased peroxidase and chitinase activities within 48 and 72 h, respectively. These results were observed for both the roots and the leaves of treated seedlings, providing evidence that T. harzianum may induce systemic resistance mechanisms in cucumber plants.

Journal Article↗

PubMed1999

Role of the Trichoderma harzianum endochitinase gene, ech42, in mycoparasitism.

The role of the Trichoderma harzianum endochitinase (Ech42) in mycoparasitism was studied by genetically manipulating the gene that encodes Ech42, ech42. We constructed several transgenic T. harzianum strains carrying multiple copies of ech42 and the corresponding gene disruptants. The level of extracellular endochitinase activity when T. harzianum was grown under inducing conditions increased up to 42-fold in multicopy strains as compared with the wild type, whereas gene disruptants exhibited practically no activity. The densities of chitin labeling of Rhizoctonia solani cell walls, after interactions with gene disruptants were not statistically significantly different than the density of chitin labeling after interactions with the wild type. Finally, no major differences in the efficacies of the strains generated as biocontrol agents against R. solani or Sclerotium rolfsii were observed in greenhouse experiments.

Blotting, Western↗

PubMed1998

The expression of genes involved in parasitism by Trichoderma harzianum is triggered by a diffusible factor.

The mycoparasite Trichoderma harzianum has been extensively used in the biocontrol of a wide range of phytopathogenic fungi. Hydrolytic enzymes secreted by the parasite have been directly implicated in the lysis of the host. Dual cultures of Trichoderma and a host, with and without contact, were used as means to study the mycoparasitic response in Trichoderma. Northern analysis showed high-level expression of genes encoding a proteinase (prb1) and an endochitinase (ech42) in dual cultures even if contact with the host was prevented by using cellophane membranes. Neither gene was induced during the interaction of Trichoderma with lectin-coated nylon fibres, which are known to induce hyphal coiling and appressorium formation. Thus, the signal involved in triggering the production of these hydrolytic enzymes by T. harzianum during the parasitic response is independent of the recognition mediated by this lectin-carbohydrate interaction. The results showed that induction of prb1 and ech42 is contact-independent, and a diffusible molecule produced by the host is the signal that triggers expression of both genes in vivo. Furthermore, a molecule that is resistant to heat and protease treatment, obtained from Rhizoctonia solani cell walls induces expression of both genes. Thus, this molecule is involved in the regulation of the expression of hydrolytic enzymes during mycoparasitism by T. harzianum.

Base Sequence↗

PubMed1998

The molecular biology of chitin digestion.

Chitinases catalyze the hydrolysis of chitin, an unbranched polymer of beta-1,4-N-acetylglucosamine. In recent years, soil-borne microorganisms that produce chitinases are considered as potential biocontrol agents against fungi and nematodes which causes diseases of agricultural crops. Chitinases also play an important physiological and ecological role in ecosystems as recyclers of chitin, by generating carbon and nitrogen sources. Many chitinases of varied organisms have been isolated and their corresponding genes cloned.

Bacteria↗

PubMed1998

Chitinolytic activity in Chromobacterium violaceum: substrate analysis and regulation by quorum sensing.

Quorum sensing control mediated by N-acyl homoserine lactone (AHL) signaling molecules has been established as a key feature of the regulation of exoenzyme production in many gram-negative bacteria. In Chromobacterium violaceum ATCC 31532 a number of phenotypic characteristics, including production of the purple pigment violacein, hydrogen cyanide, antibiotics, and exoproteases are known to be regulated by the endogenous AHL N-hexanoyl-L-homoserine lactone (HHL). In this study we show that C. violaceum produces a set of chitinolytic enzymes whose production is regulated by HHL. The chitinolytic activity was induced in strains grown in the presence of chitin as the sole carbon source and quantitated in the secreted proteins by using p-nitrophenol analogs of disaccharide, trisaccharide, and tetrasaccharide oligomers of N-acetylglucosamine. By using 4-methylumbelliferyl analogs of the same oligomers of N-acetylglucosamine as substrates for proteins separated and renatured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least six enzymes were detected: a chitobiase with high specificity to a dimeric substrate of 87 kDa, two N-acetylglucosaminidases with apparent molecular masses of 162 and 133 kDa, two endochitinases of 108 and 67 kDa, and a chitobiosidase of 56 kDa. In addition, two unidentified bands of >205 kDa were found where a tetrameric chitin derivative was used as a substrate. A pleiotropic mini-Tn5 mutant of C. violaceum (CV026) that is defective in HHL production and other quorum-sensing-regulated factors was also found to be completely deficient in chitinolytic activity. Growth of this mutant on minimal medium with chitin supplemented with culture supernatant from the C. violaceum wild-type strain or 10 microM synthetic HHL restored chitinase production to the level shown by the parental strain. These results constitute the most complete evidence so far for regulation of chitinolytic activity by AHL signaling in a gram-negative bacterium.

Acetylglucosaminidase↗

PubMed1997

Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinase-encoding gene prb1.

Transformation systems developed for Trichoderma spp. were utilized to improve the biocontrol efficiency of the mycoparasitic fungus Trichoderma harzianum by increasing the copy number of the basic proteinase gene prb1. The transformants were stable and carried from two to ten copies of prb1. High levels of expression of prb1 during fungus-fungus interaction were detected when T. harzianum and Rhizoctonia solani were confronted in vitro. In liquid cultures the proteinase was induced by cell walls of R. solani. Under greenhouse conditions, incorporation of T. harzianum transformants into pathogen-infested soil significantly reduced the disease caused by R. solani in cotton plants.

Antibiosis↗

PubMed1997

Genetic diversity and vegetative compatibility among Trichoderma harzianum isolates.

Trichoderma harzianum is the collective name of a set of asexual fungal strains which exhibit heterogeneity in genome structure, DNA sequence and behavior. Contour-clamped homogeneous field (CHEF) electrophoresis of the chromosomes of ten isolates of T. harzianum revealed six clearly distinct electrophoretic karyotypes. Of the ten isolates analyzed, four (GH12, G109, Y and YF) could be classified in a single group with identical karyotypes, while the strains T35 and 315 formed a second group. The genome size characteristic of the different isolates fell into a broad range varying from 29.6 to 56.1 Mb. Gene assignments to the resolved chromosomes showed that all genes analyzed were localized on equivalent chromosomes in the isolates belonging to the same group. Analysis of randomly amplified polymorphic DNAs from the ten isolates confirmed the classification into groups and allowed us to distinguish between isolates T35 and 315, as well as between isolates GH12, G109, Y and YF. Direct confrontation assays using isolates of the same group showed compatible interactions, whereas the same experiment carried out with isolates of different groups showed an incompatible interaction characterized by an area of cell damage. Microscopic observation of the compatible interactions showed hyphal fusions between the isolates, similar to those described for vegetative compatible groups in other fungi. The molecular karyotypes correlated well with the compatibility of the isolates. In addition, we have evaluated both electrophoretic karyotype and randomly amplified polymorphic DNAs analysis as criteria for grouping isolates within the genus according to their capacity for biocontrol of plant pathogens.

Antibiosis↗

PubMed1997

Plant resistance to fungal infection induced by nontoxic pokeweed antiviral protein mutants.

Pokeweed antiviral protein (PAP), a 29-kD protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. Transgenic plants expressing PAP are resistant to a broad spectrum of plant viruses. Nontoxic PAP mutants have been isolated by random mutagenesis and selection in yeast. One of these mutants, PAP-X, had a point mutation at the active-site (E176V) that abolished enzymatic activity, and another mutant, delta C25PAP, had a nonsense mutation near the C-terminus (W237stop) that deleted 25 C-terminal amino acids. Unlike the wild-type PAP, expression of neither mutant was toxic to transgenic plants. We show that both class I (basic) and class II (acidic) isoforms of pathogenesis-related (PR) proteins are overexpressed in transgenic plants expressing PAP and the nontoxic PAP mutants. Although PR-proteins are constitutively expressed, no increase in salicylic acid levels was detected. Homozygous progeny of transgenic plants expressing either PAP or the nontoxic PAP mutants displayed resistance to the fungal pathogen Rhizoctonia solani. These results show that expression of PAP or the nontoxic PAP mutants activates multiple plant defense pathways independently of salicylic acid and confers resistance to fungal infection. The C-terminal 25 amino acids of PAP, which are required for toxicity in vivo, are not critical for resistance to viral or fungal infection, indicating that toxicity of PAP can be separated from pathogen resistance.

Antiviral Agents↗