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PubMed1992

Antifungal proteins from plants. Purification, molecular cloning, and antifungal properties of chitinases from maize seed.

We have purified two 28-kDa chitinases, designated Chitinase A (Chit A) and Chitinase B (Chit B), from maize seeds to homogeneity and isolated cDNA clones encoding these two enzymes using an oligonucleotide probe based on an amino acid sequence of a peptide derived from Chit A. Although these two enzymes share 87% homology in their amino acid sequences, which were deduced from the nucleotide sequences of the isolated cDNA clones, they are significantly different in their biochemical and in vitro antifungal activities. When tested in vitro for antifungal activity against the growth of Trichoderma reesei, Alternaria solani, and Fusarium oxysporum, Chit A showed greater antifungal activity than Chit B. The specific activity of Chit A was determined to be 3-fold higher than that of Chit B. Chit A also had a 10-fold lower binding constant (Kd) against the substrate analogue N,N',N'',N'''-tetraacetyl chitotetrose than Chit B, indicating that the two enzyme may differ in their affinities for binding to the substrate chitin. Comparison of the amino acid sequences of maize seed chitinases with those of previously published chitinases from monocot and dicot plants indicates that maize seed chitinases have diverged significantly from other chitinases.

Alternaria↗
PubMedDate not supplied

[Germination of phytopathogenic fungi conidia].

The autoregulation of conidium germination in phytopathogenic micromycetes of the genera Fusarium, Botrytis, and Bipolaris was studied. It was shown that Trichoderma longibrachiatum was less competitive than Fusarium oxysporum after their simultaneous inoculation but inhibited the phytopathogen growth in the case of earlier introduction. In the latter case, no autoinhibition of the germination of F. oxysporum conidia occurred; moreover, cooperative effect was observed, i.e., the number of germinated F. oxysporum conidia increased with an increase in their density.

Ascomycota↗
PubMedDate not supplied

[Enzyme modification by natural chemical chaperons of microorganisms].

We demonstrated for the first time that alkylhydroxybenzenes (the d1 microbial autoregulatory factors involved in stress responses of cells) are capable of stabilizing enzymes in aqueous media and increasing their catalytic activity. The stabilizing effect of a chemical analogue of alkylhydroxybenzenes, C7-AHB, was established in in vitro studies with enzymes of microbial origin: a protease produced by Bacillus licheniformis, cellulase produced by Trichoderma viride, and alpha-amylase produced by Bacillus subtilis. This effect manifested itself in considerable extension of the temperature and pH ranges of the enzymatic activity. The modulation of the catalytic activities of the stabilized enzymes depended on the C7-AHB concentration and on the time of preincubation of the complexes obtained. We demonstrated that not only enzymes but also their polymeric substrates formed complexes with C7-AHB, and this also significantly influenced the efficiency of hydrolytic reactions. We also conducted comparative studies on the efficiency of hydrolytic reactions in systems in which the structure of enzymes and/or substrates was modified with C7-AHB.

Bacillus↗
PubMed2004

Biocontrol agents of Botrytis cinerea tested in climate chambers by making artificial infection on tomato leafs.

To reduce the use of chemical agents, that are causing damage to the environment, in the fight against Botrytis cinerea, different BCA's were tested for their possibility to control Botrytis cinerea in a biological way. In order to investigate the effectiveness of the different micro organisms and Elot-Vis, experiments were carried out in climate chambers with 5 weeks old tomato plants. Leafs on the plant were inoculated with drops of a suspension that contained spores of Botrytis cinerea. The possible antagonists that were tested in these experiments were Trichoderma harzianum (Trichodex), T. asperellum (Biofungus), T. hamatum (T382), Bacillus subtilis (Serenade and Phytovit) and Pseudomonas aeruginosa (7NSK2 and KMPCH). For all these different micro organisms the direct and the indirect influence on Botrytis cinerea was investigated. In tests where the direct influence of the antagonists was examined, the spores of the moulds or the bacteria were suspended together with spores of Botrytis cinerea and subsequently drops of this suspension were pippeted on the leafs. After a while by ideal circumstances for Botrytis cinerea the infections on the inoculated leafs were counted. For the indirect influence of the antagonists, also leafs of 5 weeks old tomato plants were inoculated with a suspension of Botrytis cinerea spores. The roots of the tomato plants that were used for testing the indirect influence were treated during there growth with a suspension of the antagonist to see if induced systemic resistance pathway (ISR) was activated. For testing the effectiveness of Elot-Vis, tomato plants were sprayed a few times with a solution of this product during their growth. Results of the climate chamber test of the plants that were treated with Elot-Vis, showed a reduction of Botrytis cinerea infections on the inoculated leafs. Biological control agents seem to be not always very effective against Botrytis cinerea. The biological control agents that are containing micro organisms are also depending on the circumstances of the environment for an optimal development. These conditions are not always that optimal to compete with Botrytis cinerea or other micro organisms that are present on the plant.

Bacillus subtilis↗
PubMedDate not supplied

[Studies of hydrolytic activity of enzyme preparations of Penicillium and Trychoderma fungi].

Enzyme preparations were isolated from the culture liquid of five mutant strains of the cellulase producer Penicillium verruculosum. The hydrolytic activities of these preparations against unbleached eucalypt cellulose was compared to that of commercial preparations of Trichoderma reesei (T. longibrachiatum). In the majority of cases, P. verruculosum enzymes provided higher yields of reducing sugars (RSs) and glucose. A correlation was found between the yield of RSs and the avicelase activity of the preparations in the reaction mixture.

Culture Media↗
PubMed2001

Microbial degradation of cellulose wastes in continuous bioreactors.

This paper reports on experiments concerning the biodegradation of cellulose wastes from wine-producing industries, as well as from cereal crops processing in agricultural farms, using cellulolytic bacterial and fungal species immobilized on radiopolymerized hydrogels. There are described three types of such bioreactors and their main features. The immobilization procedures have been achieved using the following species of microorganisms: Bacillus subtilis and B. licheniformis, from bacteria, as well as Pleurotus ostreatus and Trichoderma viride, from filamentous fungi. The immobilization matrices were prepared by radiopolymerization, using a 60Co radioactive source, designed for gamma irradiation. A consistent increasing of dry weight loss correlated with a correspondent enhancement of biodegradation rate by 10 - 20%, have revealed significant differences of metabolic activities between these immobilized bacteria and fungi on radiopolymerized hydrogels and those free cells cultures of the same microorganisms, which were grown on agar-agar gels and used as control references, in all performed experiments. Regarding the enzymatic activity of immobilized microorganisms the registered results have revealed an increasing of total reducing sugars with 20 - 30 mg g(-1). The enhancement of cellulolytic activity was higher with 0.1 - 0.3 U min(-1), in comparison with those values determined for the same process performed by the same microorganisms, used as control.

Bacillus↗
PubMedDate not supplied

[Species composition of micromycetes from sugar beet leaves, roots and rhizospheres].

Species composition of fungi, isolated from the sugar beet leaves, roots and rhizosphere, collected in Poltava and Kyiv regions has been studied. Species Alternaria, Trichoderma, Cladosporium, Acremonium, Penicillium, Fusarium, Mycelia sterilia were found in all investigated samples. Basidiomycetes were isolated from the leaf samples in separate cases.

Acremonium↗
PubMed1992

Single cell protein production from beet pulp by mixed culture.

Different mixed cultures of Trichoderma reesei and a yeast were cultivated on beet pulp (BP)-containing medium. T. reesei and Kluyveromyces marxianus offered a combination that gave high SCP yields (51%) and efficiently converted BP into proteins (39.4%). The yeast extract in the basal medium could be substituted and the BP level multiplicated from 2 to 4% upon using mixed culture of T. reesei and K. marxianus. Under these conditions the protein yields reached to a maximum value of 54% and highest efficiency of BP conversion into proteins (41.8%). The obtained protein proved to contain all essential amino acids, which compared favourably with those of the FAO guideline and soy bean oil meal.

Amino Acids↗
PubMed2005

[Screening of phytase-producing strain and its optimal solid state phytase-producing conditions].

Phytase can catalyze phytate hydrolyzed into inositol and phosphoric acid, and its application could make the phytic acid in animal feeds become more available, decrease feed cost, and at the same time, protect eco-environment. In this paper, a phytase-producing strain Trichoderma Viride LH374 was obtained through isolation and mutation, and its optimal conditions for solid state fermentation and phytase production were investigated. The results suggested that the optimal conditions of solid state fermentation were straw:rice bran ratio 8:2, medium initial pH 6.5, temperature 30 degrees C, culture duration 96 h, and moisture content 60%. Under the optimal conditions, LH374 could accumulate phytase up to 1,580 U x g(-1).

6-Phytase↗
PubMedDate not supplied

[Application of Bacillus-antagonists for biocontrol of fungi degrading raw wood].

Species composition of micromycete complexes colonizing aspen, birch, and pine wood was studied. Calculation of the Sorens-Tchekanovsky similarity coefficients showed that these complexes shared a high degree of similarity. They were dominated by the representatives of the genera Penicillium, Paecilomyces, Trichoderma, and Rhizopus. Some antagonistic bacilli inhibited growth and development of wood-decay fungi in vitro and led to the formation of spheroplasts on growing hyphae. A study of possible involvement of bacillary mycolytic enzymes in the inhibition of wood-decay fungi demonstrated selectivity of their lytic effect, which was determined by the genus and species of micromycetes and did not correlate with their relative resistance to antagonists.

Antibiosis↗
PubMedDate not supplied

[Biological preparations with different mechanism of action for protecting potato against fungal diseases].

Mycological analysis throughout the vegetation period of potato (Solanum tuberosum) made it possible to study in detail the structure of micromycete community, to determine typical dominant (frequency, more than 60%), typical common (frequency, 30 to 60%), typical rare (frequency, 10 to 30%), and casual (frequency, less than 10%) species and to estimate changes in the microorganism community caused by plant protection preparations with different mechanisms of action. It was shown that, as a result of occurrence of resistant forms, synthetic preparations against fungal pathogens of potato (such as TMTD, Ridomil gold MC, and Cupricol) were only slightly more effective than biological preparations (Trichodermin and AgroChit), with the former considerably changing the natural saprophytic mycological community. An increase in the soil pool of Trichoderma harzianum as a result of application of a biological preparation based on this antagonistic fungus correlated with its effectiveness against the soil pathogen Fusarium sp., which causes root rots. A chitosan-based elicitor preparation more effectively suppressed the development of early (Alternaria sp. and Macrosporium sp.) and late (Phytophthora sp.) blights of leaves and had a weaker effect on the soil microflora.

Alanine↗
PubMedDate not supplied

[Depolymerisation of legume seed galactomannans by Celloviridin G20x].

The depolymerisation of legume seed Galactomannans by commercial preparation "Celloviridin G20x" to produce fragments of macromolecules with unchanged monomer ratio was studied. The hydrolysis of four galactomannans in whole range of monomer ratio for this phytopolysaccharide group 1.1-5.1 was performed by enzyme preparation, which is a sum of carbohydrases (producent--Trichoderma reesei). Degree of substitution 1,4-beta-D-mannopyranose chain on C-6 position by alpha-galactose residues had influence on values of molecular mass of final reaction products. The fragments with high yelds and monomer ratio nearly of that initial polysaccharides were obtained.

Fabaceae↗
PubMedDate not supplied

[Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis].

The presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).

Alternaria↗
PubMed1991

Culture of eukaryotic cells with macro-reticulate buffers: fermentation of cellulolytic fungi.

Fermentation of fungi for large-scale production of extracellular cellulolytic enzymes requires a strict control of pH. At the lab scale, where bioreactors are not available, a culture in the exponential growth phase requires frequent manual pH adjustments. When fungi are grown in the presence of macroreticulate buffers, the culture is stable and does not require any pH control for as long as two weeks. These insoluble buffers are polyacrylamide beads (e.g., 10%T, 8%C) containing acrylamido weak acids and bases in such ratios as to unequivocally define a single pH value along the pH sale. At such pH, the macroreticulate buffers possess a strong buffering power (up to 100 milliequivalent liter-1 pH-1). In the present example, a Trichoderma sp. strain is grown in the presence of 12% beads (v/v) with an isoelectric point of 5.6, containing 100 mM of a pK 6.2 weak acrylamido base and 89 mM of a pK 4.6 weak acrylamido acid. Enzyme production (exoglucanase, endoglucanase, xylanase, beta-glucosidase) is as good as (and often better than) the control in which the pH is adjusted manually 2-3 times/day.

Buffers↗
PubMed1991

Bacterial cellulose-binding domain-like sequences in eucaryotic polypeptides.

The catalytic domain of endoglucanase II of Trichoderma reesei and the spore germination-specific polypeptide 270-11 of Dictyostelium discoideum contain amino acid sequences which share identity with the sequences of the cellulose-binding domains of several bacterial beta-1,4-glycanases. This is the first report of the presence of such sequences in eucaryotic polypeptides.

Amino Acid Sequence↗
PubMed1988

A convenient procedure for rapid release of protoplasts from Ustilago maydis.

Filtrates from cultures of Trichoderma harzianum, grown on a liquid mineral salts medium containing vitamin and trace element supplements, 0.5 g/l glucose and 2 g/l of dried residue of ethanol-extracted commercial mushrooms (Agaricus brunnescens), are a good source of enzymes for the release of protoplasts from the sporidia of Ustilago maydis. When concentrated 50-fold by (NH4)2SO4 precipitation, the enzymes liberated 95% of the protoplasts from the sporidia within 40 min.

Basidiomycota↗
PubMedDate not supplied

Isolation and liquid chromatographic determination of the cyclic peptide mycotoxin cyclosporin A from rice.

A simple method for determination and quantitation of a cyclic peptide mycotoxin, cyclosporin A, in rice is presented. Rice inoculated with Trichoderma polysporum (Link ex Pers.) was extracted with methylene chloride after 4 weeks of incubation. Cyclosporin A was isolated from extracts by using open bed gel filtration column chromatography (LH-20, acetonitrile) and monitored with thin layer chromatography and reverse phase liquid chromatography coelution with a standard. Preliminary thin layer chromatographic methods were developed. Cyclosporin A was detected by iodine and after partial acid hydrolysis by ninhydrin and UV light. A liquid chromatographic method was developed that used a reverse phase disposable cartridge cleanup and isocratic chromatography with a reverse phase octadecylsilica column and a UV detector set at 212 nm. Recovery of cyclosporin A from spiked rice samples (mg/g range) was 85%.

Chromatography, Liquid↗