P. Magro

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.

Induction of pathogenesis-related proteins in germinating wheat seeds infected with Fusarium culmorum☆

The effect of Fusarium culmorum infection during the development of wheat seedlings was investigated. The fungal infection reduced the germination of inoculated caryopses in comparison with that of control seedlings; symptoms were clearly evident on seedlings growth. At the molecular level, F. culmorum infection caused the induction of a number of pathogenesis-related (PR) proteins. Two basic isoforms of β-1,3-glucanase (PR2) and three basic isoforms of chitinase (PR3) were specifically induced upon infection, whereas several acidic and neutral glucanase and chitinase isoforms accumulate constitutively. These findings suggest that PR2 and PR3 proteins, besides their involvement in plant defense, may play a role also in the normal process of seed germination. In addition, infection by F. culmorum caused the appearance of wheatwins (PR4) and the increase of peroxidase (PR9) activity from the early stages of development of inoculated wheat seedlings. The coordinated induction of several PR-proteins with different action mechanism are part of the defense strategy that the plant activates for limiting the fungal colonization.

A basic peroxidase from wheat kernel with antifungal activity.

A basic heme-peroxidase (WP1) was purified to homogeneity from wheat (Triticum aestivum) kernels. The protein was not glycosylated and exhibited a molecular mass of 36 kDa and a pI of 8.0. The N-terminal amino acid sequence revealed a very high similarity with a wheat flour peroxidase allergen associated with bakers asthma. WPI showed indole-3-acetic acid oxidase activity in the presence of Mn2+ and phenolic cofactors. Antifungal assays performed in vitro towards phytopathogenic fungi indicated that WP1 was active in inhibiting germ tube elongation. This first report on antifungal properties of a heme-peroxidase gives experimental support to the idea that peroxidases play a defensive role against invading pathogens.

Structural and antifungal properties of a pathogenesis-related protein from wheat kernel

We have purified and characterized a protein from the water-soluble fraction of wheat kernel (Triticum aestivum cv. S. Pastore) consisting of a single polypeptide chain blocked at its N-terminus by a pyroglutamate residue; the complete amino acid sequence has been determined by automated sequence analysis performed on peptide fragments obtained by enzymatic hydrolyses of the protein. Homology studies have shown that this protein is very similar (97% sequence identity) to the previously characterized wheatwin1 as well as to other members of the pathogenesis-related (PR) proteins of class 4; in analogy with wheatwin1, we have termed this protein wheatwin2. Both wheatwin1 and wheatwin2 have specific antifungal activity toward the wide-host-range pathogenBotrytis cinerea and the wheat-specific pathogenic fungi of wheatFusarium culmorum andFusarium graminearum of groups 1 and 2. On the basis of their structural and functional properties, wheatwin1 and wheatwin2 can be classified as members of the PR4 protein family; this represents the first report concerning the presence of this kind of protein in wheat.

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.

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.

Resistance to fusarium basal rot of onion in greenhouse and field and associated expression of antifungal compounds

Greenhouse and field evaluations of onion for resistance to Fusarium basal rot caused byFusarium oxysporum f.sp.cepae were conducted on cultivars ‘Akgün 12’ and ‘Rossa Savonese’ previously described as resistant at the seedling stage. In the greenhouse experiments inoculations were carried out on seeds or soil; in the field experiments evaluation was performed on onion sets from plants grown in naturally infested soils. Akgün 12 and to a lesser extent Rossa Savonese were resistant to the disease at the bulb stage in all experiments. Results were also consistent with those obtained from a previous screening at the seedling stage. Onion sets were also extracted and fractionated by thin layer chromatography to determine their content of antifungal compounds. Extracts were characterized by the expression of distinct antifungal components, which may be involved in resistance to the pathogen.

Production of polysaccharide-degrading enzymes by Septoria Nodorum in culture and during pathogenesis

Abstract Septoria nodorum produced cell wall-degrading enzymes (CWDE) (oolygalacturonase (PG), xylanase (XY) and cellulase (CX)) both in mineral medium supplemented with wheat cell walls and in inoculated wheat leaves. The same in vitro and in vivo isoenzyme pattern for PG (pI 6.2 and 9.4) and XY (pI 5.2) was exhibited by isoelectric focusing resolution. CX was present in culture filtrates with one poorly resolved peak (pI 4.3–4.7). In infected tissues, an additional peak at pI 7.8 appeared. Some characteristics of S. nodorum polysaccharidases were compared with those of other leaf spot pathogens.

Tolerance ofsclerotinia sclerotiorum to glyceollin i, a soybean phytoalexin

Glyceollin I was fungistatic rather than fungicidal towardSclerotinia sclerotiorum. Within the mycelial mat apical cells were more vulnerable than mature cells.S. sclerotiorum removed large amounts of glyceollin from solution by a non-energy-requiring process.