Regulation of Phytoalexin Synthesis in Jackbean Callus Cultures: Stimulation of Phenylalanine Ammonia-Lyase and o-Methyltransferase.

Jackbean, Canavalia ensiformis (L.), callus tissues synthesized the phytoalexin, medicarpin (3-hydroxy-9-methoxypterocarpan), when treated with spore suspensions of Pithomyces chartarum (Berk. and Curt.) M. B. Ellis, a nonpathogen of jackbean. Medicarpin was isolated from treated callus tissue and identified by its ultraviolet and mass spectra. The minimum spore concentration found to elicit medicarpin synthesis after 26 hours was 1 x 10(5) spores/ml; levels of medicarpin in callus tissue increased linearly up to 1 x 10(7) spores/ml, indicating that the recognition sites for presumed elicitors were not saturated. Medicarpin was first detected in callus treated with 1 x 10(7) spores/ml, 6 to 12 hours after application, and the concentration reached a maximum at 48 hours, slowly declining thereafter to 72 hours. In callus treated with 3.15 mm HgCl(2), medicarpin concentrations were also maximum by 48 hours. Phenylalanine ammonia-lyase (EC 4.3.1.5) activity increased 2-fold in spore-treated callus after 36 hours. Isoliquiritigenin, daidzein, and genistein o-methyltransferase (EC 2.1.1.6) activities were increased 3- to 4-fold in treated callus. Caffeic acid and naringenin were more efficient substrates for o-methyltransferase activity than the other flavonoids or apigenin, but there was no increase in these o-methyltransferase activities in spore-treated callus. The phytoalexin response in this callus tissue culture system compares well with natural plant systems and should be an excellent system for investigating regulation of phytoalexin synthesis.

Regulation of lignin formation in reed canarygrass in relation to disease resistance.

Lignin content and enzymes involved in lignification were measured in leaf discs of reed canarygrass (Phalaris arundinacea L.) inoculated with Helminthosporium avenae and floated on water or solutions of cycloheximide (25 mug/ml). Fungal germ tubes did not penetrate localized lignified swellings, which formed beneath penetration sites, in the outer epidermal wall of discs floated on water. Within 18 hours, inoculated discs on water had higher lignin content and higher activity of the enzymes phenylalanine ammonia lyase, tyrosine ammonia lyase, hydroxycinnamate-CoA ligase and peroxidase than noninoculated discs on water. When inoculated tissues were floated on cycloheximide solutions, increases in lignin content and enzyme activities associated with lignin biosynthesis were inhibited, and the tissue was susceptible to fungal penetration. Lignin biosynthesis at the site of attempted fungal penetration may play an important role in the resistant response of reed canarygrass to leaf-infecting fungi.

Soluble and cell wall peroxidases in reed canarygrass in relation to disease resistance and localized lignin formation.

The relationship of peroxidases to an inducible disease-resistance mechanism involving lignification of leaf epidermal cell walls was studied. Reed canarygrass (Phalaris arundinacea L.) leaf discs were inoculated with Helminthosporium avenae Eidam and floated on water. In inoculated discs, the activity of soluble, ionic wall-bound and covalent wall-bound peroxidases was about twice the level of activity in noninoculated discs. The increase was attributable to increases in activity of three cathodic isoperoxidases and to the appearance of a new cathodic isoperoxidase. Peroxidase activity in cryostat microtome sections of inoculated discs was histochemically localized in the wall near the site of attempted penetration. When inoculated discs were floated on solutions of cycloheximide (25 mug/ml), increases in peroxidase activity were inhibited, and the fungus penetrated the tissue. The inhibition of peroxidase activity was related to inhibition of cathodic isoperoxidase activity. Anodic isoperoxidase activity did not show changes in response to inoculation or cycloheximide treatment.It was suggested that the resistance mechanism in P. arundinacea involves an induction of cathodic isoperoxidases in challenged tissue. These peroxidases may function in the biosynthesis of lignin at the site of attempted penetration.

Calcium Nutrition and Resistance of Alfalfa to Ditylenchus dipsaci.

Stem nematode-susceptible 'Atlantic' and resistant 'Lahontan' alfalfa seedlings, grown in sand and watered with complete nutrient solutions containing 0.75, 1.5, 3.0, 6.0, or 12.0 mM Ca/liter, were inoculated with Ditylenchus dipsaci (the stem nematode) 5-6 days after emergence. Approximately equal numbers of nematodes entered the tissues of each variety/Ca concentration within 2 days. Penetration was reduced at 12 mM Ca/liter. Reproduction during 21 days following inoculation yielded 3-fold, or greater, nematode increases in 'Atlantic' buds at all Ca concentrations, in 'Atlantic' cotyledons at the four lower concentrations, in 'Lahontan' buds at the lowest concentration and in 'Lahontan' cotyledons at the two lowest concentrations. Reproduction was lower at the higher Ca concentrations.Increased nutrient Ca concentrations resulted in increased Ca content, decreased Na and K content, and unchanged Mg content of buds and cotyledons. Accordingly, increased nutrient Ca resulted in increased divalent/monovalent cation ratios (Ca + Mg/Na + K ). Resistance to reproduction was correlated more closely with the divalent/monovalent cation ratio than with Ca content of tissue, At the four higher nutrient Ca concentrations, 'Lahontan' buds had higher ratios than 'Atlantic,' and infected buds had higher ratios than noninfected buds. Although cation balance modifies disease expression, the basic resistance mechanism remains unknown.