Gerhard A. Wolf

Dye-labelled substrates for the assay and detection of chitinase and lysozyme activity.

Abstract A carboxymethyl-substituted soluble chitin and a reprecipitated colloidal chitin derivative were covalently linked with Remazol Brilliant Violet 5R. Both conjugates are new dye-labelled substrates suitable for the assay and detection of chitinase and lysozyme activity. A reliable colorimetric microassay adapted to microtitre plates was established, based on the precipitability of carboxymethyl-chitin-Remazol Brilliant Violet 5R from buffered solutions with hydrochloric acid. Furthermore, sensitive plate-clearing assays were developed for the isolation and detection of chitinolytic microbial populations, and for the screening of pure cultures.

An oxygen-binding flavohemoprotein from Alcaligenes eutrophus

A procedure is described for the purification of a soluble flavohemoprotein from the hydrogen bacterium Alcaligenes eutrophus. The isolated protein exists as a monomer with a molecular weight of approx. 43,000. The molecule contains two prosthetic groups, 1 mol each of noncovalently bound FAD and protoheme per monomer. The absorption spectra of the protein in its ferric, ferrous-deoxy and ferrous-carboxy forms are similar to those of hemoglobins, with the exception of the flavin contribution (absorption maxima--ferric form: 395, 456, 483, 645 nm; ferrous-deoxy form: 436, 560 nm; ferrour-CO form: 423, 539, 569 nm). The flavohemoprotein when reduced by NADH in aerobic solution is capable of binding oxygen reversibly. The stable oxygenated complex exhibits absorption maxima at 414, 541, and 576 nm. The protein catalyzes the reduction of various dyes and cytochrome c by NADH.

Callase-(1,3-β-d-glucanase) activity during spring reactivation in deciduous trees

Abstract The activity of callose-degrading enzyme endo-1,3-β- d -glucanase (EC 3.2.1.39) was studied in tissue composed of cambium- and secondary phloem cells of goat willow (Salix caprea), sycamore maple (Acer pseudoplatanus), and european ash (Fraxinus excelsior) from January to May in 1989 and 1990, in relation to bud development in spring. Extracts were assayed colorimetrically for 1,3-β- d -glucanase activity by using a dye-substituted pachyman derivative as substrate. The pH optimum of glucanase activity in maple and ash was almost identical (6.0–6.5), whereas that of willow appeared to be in the acidic range (3.5–4.0). In general three seems to be a trend that glucanase activity rises in early spring consistent with it playing a role in dissolution of callose before bud break. An atypical early rise of glucanase activity in ash and maple in 1989 is probably explained by unusually high temperatures in January.

Secondary metabolite profile and phytotoxic activity of genetically distinct forms of Colletotrichum gloeosporioides from yam (Dioscorea spp.)

Highly virulent, slow-growing grey (SGG); moderately virulent, fast-growing salmon (FGS); and avirulent/weakly virulent, fast-growing grey (FGG) forms of Colletotrichum gloeosporioides have been described from yam (Dioscorea spp.), but little is known about their chemodiversity or the role of toxins in their pathogenesis. Secondary metabolite profiles in high performance tlc (hptlc) showed that the pathogenic SGG and FGS forms have a chemotype (A or B) that is distinct from the non-pathogenic FGG form (chemotype C). Crude extracts of 35-d-old Czapek-Dox yeast broth cultures of FGS and SGG isolates caused tissue necrosis on treated yam leaves but not those of FGG isolates. Extracts from uninoculated broth cultures showed no phytotoxic activity. Toxicity of the culture filtrate was not host specific and toxic substances were thermostable. Dioscorea genotypes with varying levels of resistance to anthracnose differed in their sensitivity to crude toxin extract of FGS (Cg33) and SGG (Cg25) isolates, indicating that these extracts may be useful in evaluating host resistance to anthracnose in vitro. Analysis of two toxin fractions unique to the pathogenic FGS and SGG forms using hlpc, mass spectrometry and nuclear magnetic resonance suggested the presence of a low molecular weight amide peptide. However, possibly due to low yield and the presence of impurities, the chemical structure of the compound(s) could not be fully elucidated.

Isolation of phosphodiesterase from sugar beet leaves

Abstract A phosphodiesterase (orthophosphoric diester phosphohydrolase, EC 3.1.4.1) from the leaves of sugar beets ( Beta vulgaris ) was purified 680-fold. The separation from the other plant proteins was achieved essentially by preparative electrophoresis in polyacrylamide gel. The enzyme preparation is homogeneous in disc electrophoresis under various conditions. The molecular weight is approximately 1.1·10 5 . The isoelectric point is at 3.85. The enzyme is activated by divalent ions and inhibited by EDTA. Reducing agents are also inhibitory, especially cysteine, which gives rise to degradation products that are still enzymatically active after disc electrophoresis. The phosphodiesterase is an exonuclease and produces 5′-nucleotides. The p -nitrophenyl esters of the four ribonucleotides are hydrolyzed by the enzyme at not very different rates, whereas the ester of 5′-deoxythymidylic acid is hydrolyzed considerably faster. Pyrophosphates such as ADP, ATP, and NAD are also substrates. Polyribonucleotides, RNA, and denatured DNA are also degraded, whereas native DNA is resistant. The phosphodiesterase is inhibited by its end products, the 5′-nucleotides, most strongly by 5′-adenylic acid and 5′-deoxyadenylic acid.

A soluble, dye-labelled chitin derivative adapted for the assay of krill chitinase

Abstract 1. 1. Carboxymethyl-Chitin-Remazol Brilliant Violet (CM-Chitin-RBV) was used for a colorimetric assay of chitinase activity in Antarctic krill, Euphausia superba. Comparison with a reductimetric method by end-product detection was carried out by measuring FPLC elution profiles of krill crude extracts with both assays. Both profiles matched significantly. 2. 2. Krill chitinase was highly specific to CM-Chitin-RBV. The assay was characterized by easy handling and a very high sensitivity compared to that of end-product detection. Hydrolysis of CM-Chitin-RBV by N- acetyl -β- d - glucosaminidase , β-glucosidase, β-galactosidase and N-acteyl-muraminidase was negligible. 3. 3. The enzyme characteristics of chitinase from Antarctic krill using CM-Chitin-RBV were: pHopt = 7.5, Topt = 50–55°C, Ea = 52.1 kJ · mole−1, KM = 0.07 ± 0.01 mg · ml−1.

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of loose smut of barley (Ustilago nuda)

Polyclonal antibodies were raised against mycelium from the logarithmic growth phase of a shake culture of Ustilago nuda, and a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) with biotinylated detection antibodies was developed. The detection limit of the assay was 15 ng total protein ml−1 for the homologous antigen and 50 ng ml−1 for a spore extract, respectively. Other species of Ustilago reacted with the antibodies. Cross-reactivity was highest with U. tritici. No signal was obtained with the tested isolates of Tilletia, Rhizoctonia, Pythium and Fusarium. With naturally infected barley seeds, the results of the ELISAs were always in good agreement with those obtained with the routinely used seed embryo test. However, when seeds grown from artificially inoculated florets were used, the ELISA indicated significantly higher infestation levels than the embryo test. Results of assays with halved seeds from the same lot showed that high amounts of mycelium were present in the non-embryo half. This and especially the relatively long duration of the assay suggested that the ELISA (as conducted here) may not be suitable as a routine method for analysing seed infection with U. nuda. With samples from barley seedlings grown from infected seeds the results of the immunoassay again corresponded very well with the infection level determined by staining of the seed embryo, irrespective of the mode of floret inoculation (natural or artificial). Potential fields of application of the ELISA include the early prediction of the efficacy of protection agents, e.g. in screenings for seed treatments, the elucidation of the biology of the fungus and characterisation of resistance mechanisms.

Accurate Assessment of Wheat and Triticale Cultivar Resistance to Septoria tritici and Stagonospora nodorum Infection by Biotin/Avidin ELISA

Specific and quantitative biotin/avidin-enzyme-linked immunosorbent assays (BA-ELISA) were evaluated for their ability to assess resistance of wheat and triticale cultivars to Septoria tritici (leaf blotch) and Stagonospora nodorum (leaf and glume blotch) in field trials. Using BA-ELISAs, the antigen amounts of S. tritici and of Stagonospora nodorum were measured in the flag leaf (F) and the first leaf below it (F-1) of five cultivars of triticale at Zadoks growth stage (GS) 75-80 and in 11 cultivars of wheat at GS 73-75 in 2001 and 2002. The presence of the pathogens was found to be specific to parts of the plants, cultivar, and plant species. Stagonospora nodorum was the dominant leaf blotch pathogen in triticale, while both Septoria tritici and Stagonospora nodorum occurred commonly in wheat. Close correlations were obtained between the pathogen amount measured by BA-ELISA and the percentage of necrotic leaf area in the tested cultivars. The BA-ELISA values for the tested triticale and wheat cultivars were ranked, and they correlated well with the susceptibility ratings given in the cultivar list recommended by Bundessortenamt (German Federal Office of Plant Variety), which is based on visual assessment of the leaf blotch complex caused by S. tritici and Stagonospora nodorum. The relative susceptibilities of individual wheat cultivars to both pathogens were similar. In conclusion, BA-ELISA provided for an accurate diagnosis and quantification of S. tritici and Stagonospora nodorum in infected plant tissue, and therefore can be used to assess resistance to these fungi in a disease complex in both early-stage breeding lines and field trials.

On the incorporation of32P into the various nucleic acid fractions of rust-infected primary leaves of wheat

Abstract32P administered to wheat plants for 2 h just before inoculation with stem rust, and extracted from the primary leaves 2 and 4 days later, was partly incorporated in highmolecular RNA; the specific activity of this fraction in rust-infected leaves was significantly higher than that in healthy leaves.When administered for 1 1/2 h 2 and 4 days after inoculation and extracted immediately thereafter, large amounts of32P were incorporated in a polyphosphate fraction; also in this case the specific activity of this fraction was significantly higher in rustinfected leaves than in healthy leaves. It could not be associated with a distinct RNA fraction, nor could short-living m-RNA be detected in other fractions.Samenvatting32P, gedurende 2 uur toegediend aan tarwe vlak voor inoculatie met zwarte graanroest, en 2 respectivelijk 4 dagen later geëxtraheerd, werd gedeltelijk geïncorporeerd in hoogmoleculair RNA; de specifieke activiteit van deze fractie was hoger in geïnoculeerde bladeren dan in gezonde.Indien32P gedurende 11/2 uur werd toegediend 2en 4 dagen na inoculatie en onmiddellijk daarna geëxtraheerd, werden grote hoeveelhden32P geïncorporeerd in een polyfosfaatfractie. Ook in dit geval was de specifieke activiteit van deze fractie in geïnoculeerde bladeren hoger dan in gezonde bladeren. De polyfosfaatfractie kon niet met een duidelijke RNA-fractie in verband gebracht worden; evenmin kon boodschapper-RNA in andere fracties worden aangetoond.

Soluble, dye-labelled substrates for a micro-plate assay of proteinase activity

Gelatin, collagen and casein derivatives were covalently linked with Remazol Brilliant Blue R (RBB). New dye-labelled, soluble substrates were obtained, suitable for the detection and assay of proteinase activity. A reliable colorimetric micro-plate assay was designed, based on the precipitability of gelatin-RBB, collagen-RBB and casein-RBB from buffered solutions with hydrochloric acid. Additionally, a sensitive plate-clearing assay was developed for the detection and isolation of proteolytic microorganisms from mixed populations, or for the screening of pure cultures.