Thierry C. Vrain

An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels

A two‐step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)‐PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm‐thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor‐‘sensitive’ and ‐‘insensitive’ proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology.

Effects of preplant inoculation of apple (Malus domestica Borkh.) with arbuscular mycorrhizal fungi on population growth of the root-lesion nematode, Pratylenchus penetrans

Six species of arbuscular mycorrhizal (AM) fungi (Glomus aggregatum, G. clarum, G. etunicatum, G. intraradices, G. mosseae and G. versiforme) were evaluated, in three greenhouse experiments, for their effects on reproduction of the root-lesion nematode, Pratylenchus penetrans, and growth of Ottawa 3 apple rootstock. Glomus mosseae increased total dry weights of nematode-inoculated and non-inoculated rootstock in all three greenhouse experiments, and G. intraradices increased dry weights in two of three greenhouse experiments. Plants inoculated with G. mosseae generally supported fewer P. penetrans per gram of root than plants inoculated with other AM fungi, but did not differ significantly from the controls in any greenhouse experiment. Colonization of roots by AM fungi was reduced by P. penetrans at initial inoculum densities greater than 250 nematodes/L soil. In field trials, preplant inoculation with either G. intraradices or G. mosseae increased rootstock growth and leaf concentrations of P, Mg, Zn and Cu in fumigated plots but not in non-fumigated plots, indicating that colonization by native AM fungi in non-fumigated plots may have been sufficient for adequate nutrient acquisition. The abundance of vesicles and arbuscules was greater in roots of plants inoculated with AM fungi before planting than in roots of non-inoculated plants, in both fumigated and non-fumigated plots. P. penetrans per gram of root and per 50 ml soil were significantly lower for G. mosseae- inoculated plants than for non-inoculated plants in fumigated soil but not in non-fumigated soil.

Rhizosphere colonization pattern of Agrobacterium radiobacter strain G12A, an antagonistic rhizobacterium to the potato cyst nematode Globodera pallida

Abstract The colonizing behaviour and survival of the rhizosphere bacterium Agrobacterium radiobacter G12A, an effective antagonist of the potato cyst nematode Globodera pallida , was investigated at the soil-root interface. A rifampicin resistant mutant that was used for reisolation after spray application onto tuber pieces, was quantitatively recovered 7, 14, 21, and 56 days after planting from the rhizosphere, rhizoplane, root tip, stem base, the initial tuber piece and from root-free soil mix. After 21 days A. radiobacter G12A constituted 10 to 30% of the total numbers of detectable bacteria on potato tubers and in the rhizosphere. Feeding groups of rhizoplane-colonizing microorganisms increased in density after the introduction of A. radiobacter G12A at 15 and 20 °C, but not significantly. All groups of microorganisms tested (except flourescent Pseudomonads at 25 °C) increased in the potato rhizoplane, some of them significantly, when temperature was increased from 15 °C to 25 °C.