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Work Package 1: Genetic and genomic approaches to improve protection against the main rice diseases

WP Leader: G. Valé (P1_CRA-GPG). Participants: (P1_CRA-RIS, CRA-SCV, CRA-PAV), P4, P3, P5, P8, P2.
WP1 will develop studies related to genetic resistance to the main rice pathogens affecting plant development in Italy, with the aim to identify resistance genes, provide molecular tools for MAS, detect genetic diversity and pathogenicity of the current fungal populations, and elaborate models to predict epidemics.

WP1.1 Genetic resistance to M.oryzae (rice blast) (P1).
WP1.1a) Identification and mapping of genetic loci conferring effective and durable resistance against rice blast and development of associated molecular markers for their introgression into rice breeding lines.
Two segregating F3 populations, derived from the crosses Gigante Vercelli (R) X Vialone Nano (S), and Salvo (R) x Vialone Nano (S) were developed in the framework of a previous project. Molecular analyses with molecular markers associated to several known blast resistance genes, blast infection and pedigree data from the R genotypes G.Vercelli and Salvo, support the hypothesis of sources of blast resistance still unexplored. 250 SSR markers, distributed on the 12 rice chromosomes, at an average distance of 3 Mb will be used to search polymorphisms between the parents and to construct high density genetic maps. Phenotyping for leaf and neck blast of the segregating F3 families will be conducted both using controlled inoculation with previously isolated and characterised fungal strains in growth chambers at seedling stage and with the natural inoculum strategy in field conditions. Genetic mapping of the loci/QTLs involved in blast resistance will be accomplished with the MapQTL 6 software. Genomic regions harbouring genetic determinants for blast resistance will be further enriched with additional SSR markers for fine mapping.
WP1.1b) Gene pyramiding for blast resistance genes effective against the Italian population of the blast pathogen.
Molecular markers for 4 characterized blast resistance genes (Pi-k, Pi-z, Pi-ta2, Pi-b) effective against Italian rice blast strains have been recently developed and 5 crosses between the resistance genes donors with advanced breeding lines or traditional rice cultivars are available. MAS will be used to analyse segregating progenies (at least 50 plants derived from each cross) in order to identify individual plants with pyramiding of at least two blast resistance genes. MAS will then be used to select the resistance genes during the BC generations with the advanced lines or traditional varieties.

WP1.2. Implementation of M. oryzae Italian isolate collection, epidemiology and predictive models (P4-P3).
WP1.2.a) Implementation of the M. oryzae strain collection and biomonitoring of P. grisea spores release and air diffusion to forecast rice blast disease.
Implementation of the P. grisea population collection, already deposited in the Section of Mycology, will be performed collecting samples (leaves, necks, panicles and grains) of infected rice plants during two consecutive growing seasons (June-September), particularly during maximum expected infection time (July for foliar blast; mid-August for neck blast). The presence of the pathogen in plant samples will be assessed by microscope observation and pathogen spores will be isolated from the sporulating tissues.
WP1.2.b) Simulation models are essential in quantifying the increase in productivity which can be achieved via the use of cultivars improved for their resistance/tolerance to rice blast. The blast phenotyping performed in WP1.1 together with the analysis of production data from the same genotypes will allow to create - by means of WARM and SIRBINT - virtual representations of improved cultivars with respect to the current ones. The comparison between the simulated productivity levels obtained by these virtual cultivars and by those currently available under different climatic, management and environmental scenarios, and airospora release monitoring and symptoms on plants, will allow to quantify the benefits of genotypes able to merge the high potential of the most recent cultivars with the resistance/tolerance/adaptability features that characterize some traditional varieties.

WP1.3. Fusarium complex monitoring and germplasm evaluation for resistance (P1 - P5).
WP1.3a) A Fusarium spp. collection of different Fusarium species (F. fujikuroi, F. graminearum, F. verticillioides) originated from the main Italian rice cultivation areas will be established. A population representative of the Fusarium species that, on the basis of the data obtained will be identified as the major responsible for the disease, will be morphologically and molecularly (by using PCR amplification and sequencing of informative genes) characterized with the aim of identifying the pathogen variability and presence of new pathotypes. Information obtained will be pivotal for breeding of efficient and stable resistance in rice. Representatives of these species will be used to screen the rice germplasm collection ITALORYZA (see WP4) with the aim of identify resistance sources towards the main Fusarium species responsible of root rot disease. Crossing between selected R and S genotypes will be performed to establish mapping populations to be used for disease resistance loci mapping.
WP1.3b) Fusarium-tolerant rice genotype(s) will be used for transcriptome analysis of control samples and of samples inoculated with highly pathogenic isolates using the Illumina Genome Analyzer IIx platform.
WP1.3c) Expression of key genes involved in the mycotoxin biosynthesis (P5). Italian isolates of Fusarium spp. will be evaluated for their capacity of producing fumonisins, trichothecenes and zearalenons by means of HPLC-FLD and HPLC-MS. Expression of biosynthetic genes and mycotoxin production of several isolates of the representative species will be evaluated in vitro (under different temperature, humidity and pH conditions) and in vivo (in planta studies, under different agricultural systems), to evaluate how the use of cultural systems (soil-seeded or water-seeded) and environmental parameters (temperature, relative humidity) affect the production of different mycotoxin classes and the expression level of key mycotoxin biosynthetic genes.

WP1.4 Resistance to emerging diseases (P8-P1)
WP1.4a) Bacterial foot rot, caused by Erwinia chrysanthemi pv. zeae, is one important emerging disease in rice. Bacterial strains will be isolated and characterized, and virulence levels will be evaluated on a sub-sample of the Italian rice collection.
WP1.4b) Although considered as a minor disease, helminthopsoriosis is an important rice disease affecting grain. Segregating populations of RIL (recombinant inbred lines) derived from selected crosses R x S with respect to Bypolaris oryzae, currently at RIL-F4, will be phenotyped for resistance with the aim of resistance loci mapping.

WP1.5 Realization of databases of phenotypic and genotypic data for the pathogens and the host plant (P1, P2, P5, P8).
Altogether, the phenotyping results obtained on rice genotypes will be used to realize databases dedicated to resistance sources present in the Italian rice germplasm. Similarly, results of genotyping/phenotyping obtained for M.oryzae, Fusarium, E. chrysanthemi pv. zeae, will be arranged into databases in which information about biodiversity, evolution, protocols and virulence levels will be available.