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Work Package 3: Genetic and functional diversity of rice-associated microbial communities

WP Leader P.Bonfante (P9). Participants: P9-P8-P2.
WP3 will focus on the identification and the functional analysis of the microbial communities which thrive as associated to rice plant tissues and showing different nutritional strategies (pathogens, symbionts, endophytes, saprobes). Final target: to develop innovative approaches to rice cultivation at low environmental impact and with relevant effects on rice health and stimulation of plant growth traits.


WP3.1. Molecular characterisation of the microbial communities associated to the rice rhizosphere (P2, P8, P9)
A metagenomic analysis on soils sampled from a rice field will be carried out. Four settings will be examined: rhizosphere and rice roots in aerobic and anaerobic growth conditions. A deep sequencing approach based on 454 pyrosequencing will allow to characterise bacterial and fungal communities.
Detailed experimental plan: Roots and associated rhizosphere will be sampled from three plants grown in aerobic and anaerobic conditions in three areas of the same rice field. ARISA analysis of both bacterial and fungal communities will be carried out using the capillary electrophoresis technology (AB 3730). The ARISA experiments will enable to estimate and compare the complexity of the micro-organisms communities in the four experimental settings (roots, rhizosphere in aerobic and anaerobic growth conditions). Tag-encoded 454 pyrosequencing spanning hyper-variable regions of the rRNA genes from both fungal and bacterial communities will be amplified and sequenced using the MID bar-coding technology on the Genome Sequencer (GS) FLX 454 System (Roche). Bioinformatic analysis. Bacterial and fungal rDNA obtained sequences will be quality trimmed (quality cutoff set to 20, omitting sequences <100 bp), sorted to the respective sample via the tag sequences, aligned and clustered using the pyrosequencing pipeline provided by the ribosomal database project (RDP) for bacteria and by the International Nucleotide Sequence Databases (INSD) for fungi. Operating taxonomic units (OTUs) will be classified and manually annotated. A dedicated database and sequence repository will be created facilitating the comparative analysis of the obtained metagenomic data. Differences/similarities between roots and rhizosphere microbial communities in aerobic/anaerobic conditions will be identified.


WP3.2 Endophytic bacteria thriving inside rice root and shoot tissues (P8)
This line of research intends to isolate culturable bacterial endophytes from rice grown in Italy under different conditions. Several screens will then distinguish endophytes that display significant antifungal activity in vitro and are also able to effectively colonize rice plants. The ultimate long term goal is to obtain through an intensive programme of isolation and various screens, a beneficial endophytic bacterial strain(s) which can be used as a biocontrol and biofertilizing agent to support rice cultivation in Italy.
Detailed experimental plan: 1. Isolate bacterial endophytes from rice plant tissues (shoots and roots) grown under aerobic and anaerobic conditions in Italy. 2. Perform preliminary classification of all isolated bacterial endophytes. 3. Screen isolated endophytes for in vitro anti-fungal activity. 4. The endophytes that display significant antifungal activity in vitro will be further classified. 5. Endophytes that display significant antifungal activity in vitro will be tested for colonization of rice and systemic movement within the plant. 6. The most probable very small number of endophytes which display significant anti-fungal activity and which efficiently colonize rice, will be assessed for their biocontrol and abilities as biofertilizers.


WP3.3 Investigation of the role of Arbuscular Mycorrhizae in rice roots (P9)
To understand the role of AM symbiosis in rice fields, the presence of AM fungi will be monitored during the transition from an upland towards a flooding condition. Cellular and molecular analyses will allow to verify whether changes in fungal development are related with the physiological changes already described in rice (i.e a hormonal change) during the transition from aerobiosis to anaerobiosis. Molecular analysis with quantitative RT-PCR will allow to evaluate the expression of genes currently considered functional markers of the AM symbiosis, like the phosphate and ammonium transporters, as well the expression stress-related genes.