Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 5.09

IN VIVO AND IN VITRO BIOASSAYS FOR TESTING RESISTANCE TO FUSARIUM SSP. IN WHEAT ENGINEERED WITH THE MAIZE RIP b-32

C. BALCONI*, C. LANZANOVA*, E. BALDONI*, E. CONTI*, M. CATTANEO**, E. LUPOTTO*

*) Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo

**) Istituto Sperimentale per la Cerealicoltura, Sezione di S. Angelo Lodigiano

Triticum aestivum, Fusarium, Ribosome Inactivating Protein b-32

Fusarium ssp. are widespread pathogens on cereals causing root, stem, and ear rot diseases in both temperate and semitropical areas. A maize gene, encoding a cytosolic albumin (b-32) with RIP (Ribosome Inactivating Protein) homology and biological activity, showing a potential anti-fungal role, was constitutively expressed under CaMV 35S promoter into Triticum aestivum cv. Veery, and six homozygous transgenic lines were used as experimental material.

Aim of our research is to develop a rapid and reliable screening method to evaluate the response of the transgenic wheat and other wheat genotypes upon artificial Fusarium ssp. inoculation. For this purpose, in vivo bioassays were developed to follow the progression of F. culmorum infection in inoculated wheat germinating seeds and in the subsequent developing seedlings.

F. culmorum was grown on PDA plates at 26°C until the mycelium covered the surface of the plate. To set up the optimal conditions for the bioassay, sterile seeds of the non transgenic cv. Veery, were inoculated with a conidial suspension of the fungus by adding 125, 1250 or 12500 spores/seed. Inoculated and control seeds were allowed to continue germination for 7 days. Evaluations of the response in the various genetic materials were performed according to the following parameters: i) percentage of wheat seeds with visible fungus colonization; ii) “infection score” related to the seed surface infection extent; iii) fungus colony diameter growing around infected seeds; and iv) mycelium colour intensity score. Progression of the infection on the developing vegetative tissues (radicle, coleoptyle) has also been recorded. A concentration of 125 spores/seed was useful to discriminate the response in the tested genotypes. The transgenic lines, expressing b-32 at various levels, showed differentiated and in some cases increased resistance to the Fusarium infection.

In vitro bioassays were developed to test the inhibitory activity of the maize endosperm RIP and of a commercially available plant RIP (Saporin, from Saponaria officinalis seeds), in controlling F. culmorum growth. For this purpose, known amounts of the RIP extracts were spread on agar medium and fungus radial growth was measured over time. Results indicated that a Saporin concentration of 50 µg/ml in the medium induced a significant fungus growth inhibition (≈ 40%). Current work is in progress in order to evaluate the effect of other plant commercial RIPs and to compare with purified maize b-32 on Fusarium ssp. growth, and its general effect on fungus development.