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 - 5.11

IN VIVO AND IN VITRO BIOASSAYS FOR TESTING RESISTANCE TO FUSARIUM VERTICILLIOIDES AND FOR CONTROLLING FUNGAL GROWTH WITH THE MAIZE ENDOSPERM RIP b-32

C. BALCONI, E. BALDONI, C. LANZANOVA, E. CONTI, A. MAZZOLENI, M. MOTTO, E. LUPOTTO

Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo

Zea mays, Fusarium, Ribosome Inactivating Protein b-32

Numerous Fusarium species are widespread pathogens on cereals; they can infect small grain cereals (soft and durum wheat, barley, oats, rice, triticale, sorghum, millet) and maize (mostly F. verticillioides), causing root, stem, and ear rot diseases in both temperate and semitropical areas, including all European cereal growing areas. Some Fusarium strains are also capable of producing mycotoxins which can be formed in infected plants before harvesting, or in stored grains. The occurrence of mycotoxins in cereal grains is a great concern worldwide, because their presence in feeds and foods is often associated with chronic or acute mycotoxicoses in livestock and also in humans. With this respect it is important the development of improved maize genotypes with increased resistance to F. verticillioides using traditional breeding as well as biotechnology.

The aim of our research is to develop a rapid and reliable screening method to evaluate maize genotypes for resistance to the infection of this pathogen. For this purpose in vivo bioassays were developed to follow the progression of F. verticillioides infection in inoculated maize germinating seeds and in the subsequent developing seedlings.

Maize inbred W64A and its isogenic mutant opaque-2, and inbred lines released by the Maize Station of Bergamo were used as the experimental material in order to verify if the indications obtained by the in vivo bioassay for the different genotypes, could be positively correlated to the evaluations of resistance to F. verticillioides in field experiments. F. verticillioides 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 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. For each genotype, records of the percent of maize seeds with visible fungus colonization and an “infection score”, related to the seed surface infection extent, were registered. The progression of the infection on the developing vegetative tissues (radicle, coleoptile) has been also recorded. Results indicated that the concentration of 125 spores/seed, and the second day after inoculation as timing for the infection evaluation, are the optimal conditions for the bioassay. Under these bioassay-conditions, the mutant opaque-2, lacking the endosperm Ribosome Inactivating Protein (RIP) b-32, appeared more susceptible to F. verticillioides infection both at level of germinating seeds and developing seedlings. The association of increased F. verticillioides susceptibility with RIP deficiency in opaque-2 mutant, confirmed previously reported indications about the role of b-32 in defence mechanisms against fungal pathogens. In this context, 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. verticillioides growth. For this purpose, known amounts of the RIP extracts were spread on agar medium and fungus radial growth was measured over time. Preliminary data indicated that a minimal Saporin concentration of 30-40 µg/ml in the medium was required for significant fungus growth inhibition. Current work is in progress in order to evaluate the effect of purified maize b-32 on Fusarium verticillioides growth, and its general effect on fungus development.

The work is developed within the framework of the Eu-funded project SAFEMAIZE (ICA4-CT2000-30033, Coordinator: Prof. Dave Berger, Univerity of Pretoria, SA) in FP5.