P. Marciano

Oxalic acid, cell wall-degrading enzymes and pH in pathogenesis and their significance in the virulence of two Sclerotinia sclerotiorum isolates on sunflower

Two isolates of Sclerotinia sclerotiorum, B24 (strongly virulent) and SS41 (weakly virulent), were both able to produce polygalacturonase, cellulase and xylanase in infected sunflower stems. These enzymes, even though active at their optimum pHs, were severely inhibited at pH 6·0, which was the pH of the SS41-inoculated tissues. B24-inoculated tissues, when compared with SS41-inoculated ones, were characterized by a greater amount of oxalic acid, a lack of polyphenoloxidase (PPO) activity and a lower pH (pH 4·0). Oxalic acid proved to be an inhibitor of PPO activity and its effect was particularly strong at pH 4·0. The possible significance of these factors in the virulence of the S. sclerotiorum isolates is discussed.

Polygalacturonase isoenzymes and oxalic acid produced by Sclerotinia sclerotiorum in soybean hypocotyls as elicitors of glyceollin

The polygalacturonases (PG) and oxalic acid produced by Sclerotinia sclerotiorum in infected soybean hypocotyls were investigated as elicitors of the phytoalexin glyceollin I. Purification to homogeneity through isoelectrofocusing and ion-exchange fast protein liquid chromatography revealed three endo-PG isoenzymes (PG-I, PG-II and PG-IV) and one exo-PG (PG-III) in 6-day-old etiolated soybean hypocotyls infected with the B-24 isolate of S. sclerotiorum. PG-I and PG-III, in the range of concentrations tested (0·15–1·2 reducing units ml−1), did not act as elicitors of glyceollin I synthesis. Some elicitor activity was shown by PG-II at 0·6–1·2 reducing units ml−1. PG-IV, at lower doses (0·038–0·30 reducing units ml−1), was even more effective in inducing phytoalexin synthesis. However higher concentrations of PG-IV induced tissue softening and decreased phytoalexin accumulation. PG-II and PG-IV released heat-stable elicitors from purified soybean cell walls supporting the evidence that uronides are intermediate inducers in elicitation by endo-PGs. Oxalic acid was an active elicitor of glyceollin I over the range of concentrations tested (0·31–20 mm) with the maximum at a concentration of 5 mm. The inability of oxalic acid to release uronides from purified cell walls makes it unlikely that uronide intermediate elicitors are involved in elicitation by oxalic acid.

Polygalacturonase isoenzymes produced by Sclerotinia sclerotiorum in vivo and in vitro

Abstract Sclerotinia sclerotiorum produced polygalacturonase both in Czapeks liquid medium (pectin or polygalacturonic acid as inducers) and in inoculated tissues (sunflower stems or apple fruits). The polygalacturonase complex was resolved by isoelectric focusing. In culture filtrates 1 single peak of activity was found, its isoelectric point varying depending on the inducing substrate: pI 4·8 on polygalacturonic acid medium, pI 5·1 on pectin medium. Polygalacturonase obtained from infected tissues was resolved to give a major peak at pI 8·3 and a minor one at pI 4·8. Extracts from sunflower stems, 40 h after inoculation, give a third peak, at pI 6·9. Molecular weight determinations by gel filtration showed them to be within the range 29 000 to 33 000, with the exception of isoenzyme pI 4·8 from apple, which had a molecular weight of 40 000. Isoenzymes from inoculated tissues (pI 4·8 and 8·3) and from the pectin medium (pI 5·1) were typical endopolygalacturonases but the 2 molecular forms pI 6·9 (from inoculated sunflower stems) and pI 4·8 (from polygalacturonic acid medium) were characterized by a high percentage hydrolysis and the production of free galacturonic acid after short incubation periods. The isoenzymes pI 4·8 and 8·3 from infected tissues and pI 4·8 and 5·1 from culture filtrates, caused maceration and cell death of both sunflower and apple tissues.

Characterization of two Sclerotinia sclerotiorum polygalacturonases with different abilities to elicit glyceollin in soybean

Abstract Two endo-polygalacturonase isoenzymes (PG-II and PG-IV), with masses of 34 and 30 kDa respectively, were purified from soybean hypocotyls infected by Sclerotinia sclerotiorum . The pH optimum for both isoenzymes was about 4.6, but PG-IV exhibited a broader range of pH activity. PG-IV showed a much higher affinity for pectin than did PG-II. PG-II hydrolyzed polygalacturonic acid in a more random fashion than PG-IV. Oligouronides produced by PG-II showed a higher phytoalexin elicitor activity. PG-IV produced a large degree of maceration of soybean hypocotyls releasing a significant amount of uronides. The properties of PG-II and PG-IV are discussed in relation to the different ability of the two isoenzymes to elicit glyceollin in soybean.

Variability of polygalacturonase and protein isoelectric focusing patterns in Botrytis cinerea isolates.

Acetone precipitates from culture filtrates of three isolates of Botrytis cinerea were resolved by isoelectric focusing on polyacrylamide gels to detect differences in the polygalacturonase and protein patterns. Only a few bands — four in the protein patterns and two in the polygalacturonase patterns — were common to all the isolates. Differences were also detected in polygalacturonase and protein patterns of the same isolate at different ages of culture (7, 14 and 21 d). Identical polygalacturonase patterns were obtained when isoelectric focusing was applied to an acetone precipitate either directly or after further purification by ion-exchange chromatography.

Characterization of the mycoparasite Coniothyrium minitans: comparison between morpho-physiological and molecular analyses

Eight strains of the mycoparasite Coniothyrium minitans were analysed for intraspecific variation by three different approaches: analysis of morpho-physiological characteristics; random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analysis of the genomic DNA. A high variability in colony colour, gross morphology and conidial size was found depending on strain and culture conditions. All individual strains could be distinguished using the three type of analyses. However, a close correspondence between RAPD, AFLP patterns and morpho-physiological characteristics of C. minitans strains was not observed. Our results suggest AFLP analysis constituted a more efficient and reliable tool than RAPD analysis or morpho-physiological studies to detect intraspecific variability in C. minitans and to follow its fate after application in the field.