Antifungal activity of chitosan against phytophthora infestans and activation of defence mechanisms in tomato to late blight

Chitosan (1 mg ml(-1)) reduced mycelial growth, sporangial production, release of zoospores and germination of cysts of metalaxyl resistance and sensitive Phytophthora infestans isolates. Treating tomato plants with chitosan (1 mg ml(-1)) 7 days before inoculation resulted in significant disease protection. Treatment of leaf no. 2 and 3, 7 days before inoculation induced resistance in leaf no. 4. Treatments 2-24 h after inoculation provided significant disease reduction compared with the control. Foliar application with chitosan caused small necrotic lesions on treated tomato leaves, which became visible 24 h after treatment, especially on adaxial leaf surfaces. Localized small lesions were characterized by cell death as demonstrated by irreversible uptake of Evans blue. Light and fluorescent microscopic examinations of leaf disks stained with trypan blue/Evans blue showed clusters of dead cells which were surrounded by cells exhibiting deposition of callose. In chitosan treated tomato leaves, superoxide anion (O-2(-)) generation was determined 12-36 h after treatment by nitroblue-tetrazolium staining and production of H2O2 was detected by 3,3-diaminbenzidine-4HCl staining 12-16 h after treatment. In treated tomato leaf tissues peroxidase activity increased with extending time after treatment. The highest activity was determined 48 h after treatment. Also, free and total salicylic acid contents significantly increased in chitosan treated leaves. Furthermore chitosan enhanced accumulation of pathogenesis related-proteins such as beta-1,3-glucanase, chitinase and PR14 in treated and upper untreated tomato leaves. The studies with chitosan against tomato late blight suggested that chitosan displays dual effects: (a) direct interference in developmental stages of P. infestans and (b) by lesion formation, leading to disease resistance mechanisms.