Rhamnogalacturonase (RGase) is a new fungal enzyme which degrades the highly branched regions of apple fruit cell wall pectin by cleaving the glycosyl linkage between rhamnosyl and galacturonosyl residues (Schols et al., 1990. Carhohydr. Res. 206:105.). This enzyme, if present in fruit, could play a significant role in fruit softening. Partial purification of RGase was accomplished from a fungal enzyme preparation (Pectinex Ultra SP-L, NOVO Ferment) produced from Aspergillus niger. The crude enzyme hydrolyzed chelator-soluble pectin from red ripe tomato fruit. Methylation linkage analysis of the product suggested that an increase in terminal-rhamnosyl residues accompanied pectin hydrolysis, indicative of RGase activity. Cross-linked alginate, hydroxyapatite, and DEAE-Sephadex chromatography were used to partially purify RGase. Polygalacturonase was efficiently removed using the alginate column. Crude pectin obtained from mature-green tomato fruit cell wall by extracting with 0.5 M imidazole buffer (pH 7) and 50 mM Na-carbonate was incubated with pure polygalacturonase and the residue hydrolyzed with 0.1 N trifluoroacetic acid. This modified pectin was used as a substrate to investigate the presence of RGase in tomato and other fruit.
Jongkee Kim and Kenneth C. Gross
Won Jun, Soo-Seong Lee and Jongkee Kim
Three inoculation methods, including cutting of a leaf, drenching, and point inoculation, were compared in an effort to screen the susceptibility of Chinese cabbage to soft rot disease caused by Erwinia carotovora subsp. carotovora. Three- to 4-week-old seedlings from 10 lines of cabbage with 16-h-old bacterial culture were routinely used. Inoculated seedlings were kept at 25.0 ± 10.0 °C for 48 h with saturated water vapor using a plastic tunnel in a greenhouse. Sixty-day-old mature plants were produced and inoculated in a greenhouse. Severity of symptoms, which were observed from percentage of plant infected was scored as from 1 to 9, representing resistant to susceptible, respectively. The correlation between seedlings and mature plants from ten lines was evaluated among the three different inoculation methods. Point inoculation gave the most significant correlation (r = 0.843, P < 0.05) between seedlings and mature plants. A good correlation was also observed between point inoculation of seedlings and drenching of mature plants (r = 0.609, P < 0.05). Cutting of a leaf of seedlings was also correlated with point inoculation of mature plants (r = 0.609, P < 0.05). This method provides the advantage of being able to keep the experimental plant alive, as only one leaf is detached. The point inoculation method is simple and relatively sensitive, so it could be used for large-scale screening for this bacterial soft rot disease. From three different evaluation assays, it was concluded that the breeding lines, C3-28, C3-29 from Cornell Univ. (Geneva, N.Y.) and the cultivar Kweonsim319 were relatively resistant to bacterial soft rot, while the Cornell breeding line CC-25 and the `Rang-no' cultivar were relatively susceptible.