A. Sivan

Biodegradation of polyethylene by the thermophilic bacterium Brevibacillus borstelensis

Aim:  To select a polyethylene‐degrading micro‐organism and to study the factors affecting its biodegrading activity.

Degradation of Fungal Cell Walls by Lytic Enzymes of Trichoderma harzianum

SUMMARY: In in vitro tests, two strains of Trichoderma harzianum failed to parasitize colonies of Fusarium oxysporum f. sp. vasinfectum and F. oxysporum f. sp. melonis. However, these strains were strongly mycoparasitic on Rhizoctonia solani and Pythium aphanidermatum. When grown in liquid cultures containing laminarin, chitin or fungal cell walls as sole carbon sources, both strains of T. harzianum released, 3-β-glucanase and chitinase into the medium. Higher levels of these enzymes were induced in strain T-203 than in T-35 by hyphal cell walls of F. oxysporum. When the lytic enzymes produced by T-35 were incubated with hyphal cell walls of the test fungi, more glucose and N-acetyl-d-glucosamine was released from cell walls of R. solani and Sclerotium rolfsii than from those of F. oxysporum. Treatment of F. oxysporum cell walls with 2 m-NaOH, protease or trypsin prior to their incubation with the lytic enzymes of T. harzianum significantly increased the release of glucose and N-acetyl-d-glucosamine. The effect of these treatments on R. solani and S. rolfsii cell walls was much lower. These results suggest that proteins in the cell walls of F. oxysporum may make these walls more resistant than those of R. solani or S. rolfsii to degradation by extracellular enzymes of T. harzianum.

Stable chloroplast transformation of the unicellular red alga Porphyridium species.

Red algae are extremely attractive for biotechnology because they synthesize accessory photosynthetic pigments (phycobilins and carotenoids), unsaturated fatty acids, and unique cell wall sulfated polysaccharides. We report a high-efficiency chloroplast transformation system for the unicellular red microalgaPorphyridium sp. This is the first genetic transformation system for Rhodophytes and is based on use of a mutant form of the gene encoding acetohydroxyacid synthase [AHAS(W492S)] as a dominant selectable marker. AHAS is the target enzyme of the herbicide sulfometuron methyl, which effectively inhibits growth of bacteria, fungi, plants, and algae. Biolistic transformation of synchronized Porphyridium sp. cells with the mutant AHAS(W492S) gene that confers herbicide resistance gave a high frequency of sulfomethuron methyl-resistant colonies. The mutant AHAS gene integrated into the chloroplast genome by homologous recombination. This system paves the way for expression of foreign genes in red algae and has important biotechnological implications.

IMPROVED RHIZOSPHERE COMPETENCE IN A PROTOPLAST FUSION PROGENY OF TRICHODERMA HARZIANUM

Summary: The level and pattern of rhizosphere competence of a strain of Trichoderma harzianum (1295-22) derived from fusing protoplasts of auxotrophic mutants of the prototrophic strains T12 and T95 were studied and compared with those of the original strains. Colonization of the rhizosphere by the three strains was tested after treating seeds of cotton and maize with conidia and planting them in soil at a constant moisture content. Propagules of the fungi were removed by a washing technique, Trichoderma spp. were isolated by plating serial dilutions on a selective medium, and individual strains were identified by their characteristic growth on differential media. Both strains T12 and T95 colonized the entire length of maize roots, but strain 1295-22 was more effective in colonizing the middle sections of the roots than either parental strain. All strains colonized cotton roots more poorly than maize roots; strains T12 and T95 were not detected on some root segments of this crop. Strain T95 was, however, found on the root tip, while T12 was absent from this root portion. Conversely, strain 1295-22 colonized all root sections of this crop, and its population levels were higher in the middle root portions than those of either parental strain.

Control of Rhizoctonia solani fruit rot of tomatoes by Trichoderma harzianum Rifai

Abstract Application of Trichoderma harzianum, to soil or by coating tomato fruits, reduced Rhizoctonia solani fruit rot by up to 43% and 85%, respectively, under laboratory conditions. When mixed with naturally infested soil, Trichoderma reduced R. solani inoculum potential by 86% in field trials. It also significantly reduced fruit rot by 27–51%.

TRANSFORMATION OF TRICHODERMA SPP. WITH PLASMIDS CONFERRING HYGROMYCIN B RESISTANCE

Several plasmids with different promoter sequences, all containing the HygB gene that confers resistance to the antibiotic hygromycin B, were evaluated for their ability to transform Trichoderma viride strain T 105-227 Nic-. Plasmid pH IB, which contains a promoter sequence from Cochliobolus heterostrophus, was found to give the largest number of putative transformants. Transformation of T. harzianum strains T12-2 His- and T95-1 Lys- was attempted, and the time at which transformed protoplasts were exposed to hygromycin B was found to be critical. If hygromycin B was added before protoplasts had regenerated for at least 6 h, no transformants were obtained. However, if regeneration proceeded for more than 12 h, many nontransformed protoplasts formed thalli. Putative transformants were heterokaryotic. Even if thalli were grown in the presence of hygromycin B, the number of transformed nuclei was low; only 0.5 to 0.8% of conidia from putative transformants were capable of producing thalli in the presence of hygromycin B. Subcultures derived from single hygromycin B-resistant conidia were stable. Southern analysis indicated that the plasmid was integrated into the genome in either tandem repeat pattern or was single copy insertion.

Potential production of protoplasts from Porphyridium sp. using an enzymatic extract of its predator Gymnodinium sp.

Abstract Production of biochemicals from red algae will become an agro-industrial reality only after improvement of strain through genetic manipulation has been achieved. In the absence of sexual reproduction, preparation of protoplasts is a prerequisite for genetic improvement of new strains. Although preparation of protoplasts from plant cells is a common technique, its application in red algae was limited. The unicllular alga Porphyridium sp. is encapsulated in a sulfated polysaccharide, the structure of which is still not fully known. A crude extract of a dinoflagelate Gymnodinium, a natural predator of Porphyridium cells in open cultures, was found to degrade Porphyridium sp. polysaccharide enzymatically. Porphyridium cells treated with the crude Gymnodinium extract were exposed to various osmotic media (0–1·5 m sucrose), and their volume was measured. Volume increase was observed in diluted sucrose solutions up to 0·175 m . While further dilution of the external osmoticum to 1·0 m had little effect, dilution to 0·0 m (distilled water) led to cell rupture. Elevated concentrations of external osmoticum resulted in shrinkage of the treated cells. Such osmotic behavior indicates exposure of the cells and thus cleavage of the capsule. The treatment did not affect the viability of the cells, as evidenced by fluorescein diacetate (FDA) fluorescence, nor did it affect the respiration rate, but it did lower the photosynthetic rate to some extent. The growth curves for the treated cells exhibited a longer lag time than in the non-treated controls. Lowering the NaCl content in the growth medium resulted in a further increase in the lag time of the treated cells. These results indicate that the treatment lowers the ability of Porphyridium cells to divide. Ability to divide is eventually recovered with time, the recovery apparently depending upon the external osmoticum. The results indicate that Gymnodinium crude extract degrades Porphyridium cell wall and thus can be used for protoplast production.