Raising antibodies against epoxyscillirosidine, the toxic principle contained in Moraea pallida Bak. (Iridaceae), in rabbits.

Moraea pallida Bak. (yellow tulp) poisoning is the most important plant cardiac glycoside toxicosis in South Africa. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. The aim was to investigate the potential to develop a vaccine against epoxyscillirosidine. Epoxyscillirosidine, proscillaridin and bufalin, were successfully conjugated to hen ovalbumin (OVA), bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH). There was a low immune response following vaccination of adult male New Zealand White rabbits with epoxyscillirosidine-OVA (n = 3) and OVA (n = 3) using Freund's adjuvant in Trial (T) 1. The immune response improved significantly in T2 following doubling of the dose to 0.8 mg/rabbit and changing the adjuvant to Montanide. In T3, the rabbits (n = 15), allocated into 5 equal groups, vaccinated with proscillaridin-BSA, bufalin-BSA, epoxyscillirosidine-KLH, epoxyscillirosidine-BSA and BSA respectively, using Montanide adjuvant, developed antibodies against the administered immunogens, with epoxyscillirosidine-KLH inducing the highest immune response. Proscillaridin and bufalin antibodies cross-reacted with epoxyscillirosidine in an enzyme linked immunosorbent assay. The conjugation methodology will be adjusted in the future to target optimal conjugation efficiency. Additional vaccination will be conducted in search of neutralizing antibodies against the yellow tulp toxin. The cross-reactivity of proscillaridin and bufalin antibodies with epoxyscillirosidine could be studied in future to explore the potential to prevent yellow tulp poisoning.

The NPR1 homolog GhNPR1 plays an important role in the defense response of Gladiolus hybridus.

KEY MESSAGE: GhNPR1 shares similar functions as Arabidopsis NPR1 . Silencing of GhNPR1 in Gladiolus results in an enhanced susceptibility to Curvularia gladioli. We propose that GhNPR1 plays important roles in plant immunity. Gladiolus plants and corms are susceptible to various types of pathogens including fungi, bacteria and viruses. Understanding the innate defense mechanism in Gladiolus is a prerequisite for the development of new protection strategies. The non-expressor of pathogenesis-related gene 1 (NPR1) and bzip transcription factor TGA2 play a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). In this study, the homologous genes, GhNPR1 and GhTGA2, were isolated from Gladiolus and functionally characterized. Expression of GhNPR1 exhibited a 3.8-fold increase in Gladiolus leaves following salicylic acid treatment. A 1332 bp fragment of the GhNPR1 promoter from Gladiolus hybridus was identified. Inducibility of the GhNPR1 promoter by SA was demonstrated using transient expression assays in the leaves of Nicotiana benthamiana. The GhNPR1 protein is located in the nucleus and cytomembrane. GhNPR1 interacts with GhTGA2, as observed using the bimolecular fluorescence complementation system. Overexpression of GhNPR1 in an Arabidopsis npr1 mutant can restore its basal resistance to Pseudomonas syringae pv. tomato DC3000. Silencing of GhNPR1, using a tobacco rattle virus-based silencing vector, resulted in an enhanced susceptibility to Curvularia gladioli. In conclusion, these results suggest that GhNPR1 plays a pivotal role in the SA-dependent systemic acquired resistance in Gladiolus.

In vitro selection for Fusarium wilt resistance in Gladiolus.

Cormels pieces of four Fusarium susceptible Gladiolus cultivars (Friendship, Peter Pears, Victor Borge and Novalux) formed friable calli when cultured in vitro on Murashige and Skoog basal medium containing various concentrations of auxin and cytokinin. The friable calli established cell suspensions. Plantlet regeneration was obtained from the control callus, control cell suspension derived callus and in vitro selected Fusarium oxysporum Schlecht. resistant cell-lines of Friendship. The in vitro cormlets showed 85-95% germination after breaking dormancy of 8 weeks at 4 degrees C. Cell suspensions of all four Gladiolus cultivars were found to be highly sensitive to fusaric acid. Gradual increase in fusaric acid concentrations to the cell-suspension cultures decreased cell growth considerably. One albino plant was found from the second generation of the in vitro selected cell line of Friendship. The albino plant was found to be highly susceptible to F. oxysporum. The cormlets of all in vitro selected cell lines of Friendship were inoculated with a conidial suspension of the F. oxysporum before planting and were also sprayed with the same spore suspension for further characterization when the height of plants was about 6 cm. The four selected cell lines showed the same response whether or not they were inoculated with conidia of the F. oxysporum. Plantlets of all of the selected cell lines exhibited significant growth as compared with the control after application of conidia of the F. oxysporum.