Trichoderma Harzianum Strain Research Articles

Growth Promotion of Highbush Blueberry by Fungal and Bacterial Inoculants

The highbush blueberry cultivar Bluecrop was inoculated with potential plant growth-promoting (PGPR) candidates, including bacterial inoculants Pseudomonas fluorescens (Migula) (strains Pf 5, PRA 25, 105, or 101), Bacillus pumilus (Mayer and Gottheil) (strain T4), Pseudomonas corrugata (Roberts and Scarlett) (strain 114), and fungal isolates Gliocladium virens (Miller et al., Von Arx) (strain Gl.21) and Trichoderma harzianum (Rifai) (strain T 22). Addition of G. virens to pasteurized soil increased leaf area and the number of leaves produced in a 4-month growth period, as well as shoot content of P, Zn and Cu in 1997. Treatment with P. fluorescens Pf 5 increased leaf area and stem diameter. In nonpasteurized soil, plants inoculated with G. virens had greater leaf area, stem diameter, shoot and root dry weight, and more leaves per plant. These results demonstrate the potential of G. virens for increasing growth when used to inoculate blueberry plants in the nursery or at transplanting.

Induction and accumulation of PR proteins activityduring early stages of root colonizationby the mycoparasite Trichoderma harzianum strain T-203

The biochemical nature of the interaction between the antagonistic fungus Trichoderma harzianum strain T-203 and cucumber roots was studied during the early stages of root colonization by the fungus. Pathogenesis related (PR) proteins of the plant and enzyme activity of the fungus following the penetration and colonization of the roots by T. harzianum were explored up to 72 h post-inoculation. Scanning electron microscopy (SEM) revealed typical fungal structures previously associated with mycoparasitic interactions of T. harzianum strains during biological control. These included hyphal coiling and appressoria formation. Compared to untreated control, cucumber roots treated with T. harzianum T-203 exhibited higher activities of chitinase (EC 3.2.1.14), β-1,3-glucanase (EC 3.2.1.6), cellulase (EC 3.2.1.4) and peroxidase (EC 1.11.1.7), up to 72 h post-inoculation. Plants treated with a chemical inducer of the plant defence response, 2,6-dichloroisonicotinic acid (INA) displayed responses that were similar but not identical to those of plants inoculated with T. harzianum. In vivo staining of chitinase activity in fresh root sections allowed the localization of the activity in roots treated with either T. harzianum T-203 or INA. The formation of fluorescent products mainly in intercellular spaces of the induced roots provided evidence for the involvement of the plant defence system. In addition to its well-recognized mycoparasitic nature, it is suggested that Trichoderma’s association with roots reduce root disease through activation of the plant’s defence response.

Hydrolysis of xylans by enzyme systems from solid cultures of Trichoderma harzianum strains.

Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28 degree C in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ss-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20. 45 cp. The efficiency of delignification was 33%.

A new xylanase from a Trichoderma harzianum strain.

A new xylanase (XYL2) was purified from solid-state cultures of Trichoderma harzianum strain C by ultrafiltration and gel filtration. SDS-PAGE of the xylanase showed an apparent homogeneity and molecular weight of 18 kDa. It had the highest activity at pH 5.0 and 45 degrees C and was stable at 50 degrees C and pH 5.0 up to 4 h xylanase. XYL2 had a low Km with insoluble oat spelt xylan as substrate. Compared to the amino acid composition of xylanases from Trichoderma spp, xylanase XYL2 presented a high content of glutamate/glutamine, phenylalanine and cysteine, and a low content of serine. Xylanase XYL2 improved the delignification and selectivity of unbleached hardwood kraft pulp.

Ionophoric activity of the antibiotic peptaibol trichorzin PA VI: a 23Na- and 35Cl-NMR study

Trichorzin PA VI (Ac Aib 1 Ser Ala Aib Iva Gln Aib Val Aib Gly 10 Leu Aib Pro Leu Aib Aib Gln Pheol 18) is one of the seven main peptaibols forming the natural antibiotic 18-residue peptide mixture biosynthesised by a Trichoderma harzianum strain. Trichorzins exhibit antimycoplasmic activity resulting from membrane permeability perturbations. The membrane permeabilisation process by trichorzin PA VI has been examined in egg yolk phosphatidylcholine large unilamellar vesicles (LUV) and under conditions of ionic equilibrium by 23Na- and 35Cl-NMR experiments conducted in the presence of a chemical shift reagent and a relaxation agent, respectively. In such conditions, trichorzin PA VI exchanges both cations and anions across the vesicle bilayers, indicating the absence of ion- and charge-selectivity, in contrast to antibiotic ionophores, such as monensin or nigericin; the Na + exchange is not influenced by the ionic strength. The kinetics of the Na + exchange have been found to be third to fourth order with respect to the peptide concentration. The permeabilisation process of liposomes has been shown to be due to the formation of aggregates of three to four helical peptide monomers arranged into a supramolecular complex including presumably lipid molecules and forming a badly-defined pore in the bilayer. The major mechanism by which ions may exchange through the bilayer involves a long-lasting opening of the pores allowing complete exchange of the internal and external media in an ‘all or nothing mode’.

Ecological Studies of Transformed Trichoderma harzianum Strain 1295–22 in the Rhizosphere and on the Phylloplane of Creeping Bentgrass

ABSTRACT A beta-glucuronidase (GUS) reporter gene and a hygromycin B (hygB) phosphotransferase gene were integrated separately into the Trichoderma harzianum strain 1295-22 genome, using biolistic transformation. The mycelial growth and biocontrol ability of the transformed strains did not differ from that of the original strain. The transformed Gus(+)-kanamycin-resistant (Gus(+)Kan(R)) strains were used to monitor growth and interactions with Rhizoctonia solani on creeping bentgrass plants. The hygB-resistant (hygB(R)) strains were used to selectively recover strain 1295-22 from the rhizosphere soil and phylloplane of creeping bentgrass after spray applications. The population levels of two hygB(R) strains and the original strain were very similar for all treatments. All three strains persisted for the duration of the experiment (28 days) in both the rhizosphere soil and on leaves, although population levels declined somewhat over the course of the experiment in unautoclaved soils. In this study, the results demonstrated that hygB(R) strains remained dominant over time when assayed on Trichoderma-selective medium containing hygB. The hygB(R) strains were not displaced by strains that colonized untreated plants. Microscopic observation showed that the Gus(+)Kan(R) strains colonized the rhizoplane, seed coat, and phylloplane of creeping bentgrass. These results supported our earlier observation that strain 1295-22 was rhizosphere and phyllo-plane competent. Interactions between T. harzianum and R. solani were readily observed in situ and changed over time. Two types of reactions were found in these experiments. In the first type, sections of hyphae of R. solani near the hyphae of T. harzianum appeared damaged, and the pathogen appeared necrotic when viewed with a microscope. The second type, observed less frequently than the first type, was typical of myco-parasitism. The findings in this study provide new insight into the interactions between R. solani and T. harzianum, providing a basis for future research.

Harzianin HB I, an 11-residue peptaibol from Trichoderma harzianum: isolation, sequence, solution synthesis and membrane activity

Harzianin HB I is a minor component of the peptaibol mixture biosynthesized by a Trichoderma harzianum strain. It is isolated by a multi-step chromatography procedure, including HPLC; its sequence has been elucidated by LSIMS and two-dimensional 1H NMR experiments. This 11-residue peptaibol is an analogue of the 14-residue peptaibol harzianin HC IX, resulting from the deletion of the tripeptide Aib-Pro-Ala. The CD and NMR data (NOE data and amide proton temperature coefficients) are similar to those of HC IX, suggesting a right-handed helix-type conformation for the two peptides. Harzianin HB I was synthesized by the solution-phase method using BOP as coupling reagent. The membrane properties examined on liposomes are compared with those of other known peptaibols.

Isolation and sequence analysis of the peptide antibiotics trichorzins PA from Trichoderma harzianum

Trichorzins PA are new members of the class of natural Aib-containing peptides, which are known under the common name peptaibols. They are produced by a Trichoderma harzianum strain and exhibit significant activity against mycoplasmas. From the natural peptide mixture, seven trichorzins PA (PA II and IV–IX) have been isolated by a procedure employing different chromatography steps and a final purification by reversed-phase HPLC. The amino acid sequence elucidation involved a combination of liquid secondary-ion mass spectrometry (LSIMS) and two-dimensional 1 H and 13 C NMR spectroscopy. Trichorzins PA are 18-residue peptaibols which differ mainly from other, related peptides by the C-terminal amino alcohol which is either tryptophanol or phenylalaninol. They adopt a helical structure.

Production of β-Xylosidase Activity by Trichodermaharzianum Strains

Nine Trichoderma harzianum strains were screened for beta-xylosidase activity when grown in solid-state cultures on media containing wheat bran as the carbon source. All strains produced beta-xylosidase activity, the most active being in extracts of cultures of T. harzianum strain 4. A beta-xylosidase was purified by ammonium sulfate precipitation, ultrafiltration, gel filtration, and ion exchange chromatography from solid-state cultures of T. harzianum strain C. Enzyme preparations yielded a single band when stained for protein following eletrophoresis. The molecular weight value, calculated following SDS-PAGE, was determined to be 60 kDa. beta-Xylosidase was most active at pH 4.0-4.5 and 70 degrees C. This enzyme had a Km value of 0.053 mM. The phenol-sulfuric acid method detected the presence of a small amount of carbohydrate in the purified enzyme preparation. beta-Xylosidase was active against some p-nitrophenylglycosides. The enzyme was inactive against xylan and PNPG. beta-xylosidase activity was inhibited by xylose and SDS. Iodoacetamide, dithiothreitol, gluconolactone, glucose, and mercuric chloride failed to inactivate this enzyme's activity. A synergistic effect was observed when beta-xylosidase from T. harzianum strain C and beta-xylanase from Aspergillus fumigatus were incubated with pretreated arabinoxylan.

Fate of transformed Trichoderma harzianum in the phylloplane of tomato plants

AbstractA propiconazole‐resistant Trichoderma harzianum strain with high phylloplane survival capability was transformed with the E. coli hygromycin B phosphotransferase gene (hph), coding for hygromycin B resistance. Four transformants were analysed for survival ability on the phylloplane of tomato plants grown under glasshouse conditions in comparison with their prototype and a yellow, hygromycin B‐sensitive mutant. Over 2 weeks, the four transformants showed higher survival rates in comparison with the wild‐type strain. The yellow mutant TF3/973 did not significantly differ in survival from the transformants. Both hygromycin B resistance and mitotic stability of transformants were evaluated during growth in vitro and after reisolation from tomato phylloplane. Hybridization patterns with the complete plasmid indicated that all four transformants were mitotically stable after several rounds of vegetative growth without selective pressure and during 2 weeks on tomato plants. None of the transformants had lost the ability to grow in the presence of both propiconazole and hygromycin B after growth under the same conditions. The results are discussed in relation to risk assessment of the release of transgenic fungi.